NUCLEIC ACIDS COMPRISING FORMULA (NuGlXmGnNv)a AND DERIVATIVES THEREOF AS IMMUNOSTIMULATING AGENT/ADJUVANT

ABSTRACT

The present invention relates to nucleic acids of the general formula (I): (N u G l X m G n N v ) a  and derivatives thereof as an immunostimulating agent/adjuvant and to compositions containing same, optionally comprising an additional adjuvant. The present invention furthermore relates to a pharmaceutical composition or to a vaccine, each containing nucleic acids of formula (I) above and/or derivatives thereof as an immunostimulating agent, and optionally at least one additional pharmaceutically active component, e.g. an antigenic agent. The present invention relates likewise to the use of the pharmaceutical composition or of the vaccine for the treatment of cancer diseases, infectious diseases, allergies and autoimmune diseases etc. Likewise, the present invention includes the use of nucleic acids of the general formula (I): (N u G l X m G n N v ) a  and/or derivatives thereof for the preparation of a pharmaceutical composition for the treatment of such diseases.

This application is a continuation of U.S. application Ser. No.12/672,442, filed Oct. 14, 2011, which is a national phase applicationunder 35 U.S.C. §371 of International Application No. PCT/EP2009/000546,filed Jan. 28, 2009, which claims benefit of European Application No. EP08001827.8, filed Jan. 31, 2008. The entire text of each of theabove-referenced disclosures is specifically incorporated herein byreference.

The present invention relates to nucleic acids of the general formula(I): (N_(u)G_(l)X_(m)G_(n)N_(v))_(a) and derivatives thereof as animmunostimulating agent/adjuvant and to compositions containing same,optionally comprising an additional adjuvant. The present inventionfurthermore relates to a pharmaceutical composition or to a vaccine,each containing nucleic acids of formula (I) above and/or derivativesthereof as an immunostimulating agent, and optionally at least oneadditional pharmaceutically active component, e.g. an antigenic agent.The present invention relates likewise to the use of the pharmaceuticalcomposition or of the vaccine for the treatment of cancer diseases,infectious diseases, allergies and autoimmune diseases etc. Likewise,the present invention includes the use of nucleic acids of the generalformula (I): (N_(u)G_(l)X_(m)G_(n)N_(v))_(a) and/or derivatives thereoffor the preparation of a pharmaceutical composition for the treatment ofsuch diseases.

In both conventional and genetic vaccination, the problem frequentlyoccurs that only a small and therefore frequently inadequate immuneresponse is brought about in the organism to be treated or inoculated.For this reason, so-called adjuvants are frequently added to vaccines orpharmaceutically active components, that is to say substances orcompositions that are able to increase and/or influence in a targetedmanner an immune response, for example to an antigen. For example, it isknown that the effectiveness of some injectable medicinal activeingredients can be improved significantly by combining the activeingredient with an adjuvant which is capable of influencing the releaseof the active ingredient into the host cell system and optionally itsuptake into the host cells. In this manner it is possible to achieve aneffect that is comparable to the periodic administration of many smalldoses at regular intervals. The term “adjuvant” conventionally refers inthis context to a compound or composition that serves as a carrier orauxiliary substance for immunogens and/or other pharmaceutically activecompounds. Typically, the term “adjuvant” is to be interpreted in abroad sense and refers to a broad spectrum of substances or stratagerms,that are able to increase the immunogenicity of antigens incorporatedinto or coadministered with an adjuvant in question. Adjuvantsfurthermore may be divided, without being limited thereto, into immunepotentiators, antigenic delivery systems or even combinations thereof.

A number of compounds and compositions have been proposed as adjuvantsin the art, for example Freund's adjuvant, metal oxides (aluminiumhydroxide, etc.), alum, inorganic chelates or salts thereof, variousparaffin-like oils, synthetic resins, alginates, mucoids, polysaccharidecompounds, caseinates, as well as compounds isolated from blood and/orblood clots, such as, for example, fibrin derivatives, etc. However,such adjuvants in most cases produce undesirable side-effects, forexample skin irritation and inflammation at the site of administration.Even toxic side-effects, in particular tissue necroses, are alsoobserved in some cases. Unfortunately, in most cases these knownadjuvants bring about only inadequate stimulation of the cellular immuneresponse, because only B-cells are activated.

Compounds isolated from animals, such as, for example, gelatin, aregenerally not suitable as adjuvants for the purpose ofimmunostimulation. Although such compounds usually do not exhibit anegative effect on the host organism or the host cells in question, theytypically migrate too rapidly from the injection site into the hostorganism or into the host cells, so that the properties generallydesired for an adjuvant, such as, for example, delayed release of anactive ingredient optionally injected together with the adjuvant, etc.,are seldom achieved. Such rapid distribution can, in some cases, becounteracted with tannins or other (inorganic) compounds. The metabolismof such additional compounds and their whereabouts in the body has notbeen fully explained, however. In this case too, therefore, it isreasonable to assume that these compounds accumulate in the debris andthus considerably interfere with the filtration mechanisms, for examplethe kidney, liver and/or spleen cells. Also, the property of gelatin ofswelling when administered parenterally can lead to unpleasantside-effects under in vivo conditions, such as, for example, swelling,in particular at the site of administration, and to a feeling ofillness.

In the case of compounds isolated from blood and/or blood clots, suchas, for example, fibrin derivatives, etc., immunostimulating effectshave typically been demonstrated. However, most of these compounds, whenadministered as adjuvants, are not suitable for that purpose because oftheir side-effects on the immune system (which occur in parallel withthe required immunogenic properties). For example, many of thesecompounds are categorised as allergenic and in some circumstances leadto an excess reaction of the immune system which far exceeds the desireddegree. These compounds are therefore likewise unsuitable as adjuvantsfor immunostimulation for the mentioned reasons.

Accordingly, it is a first object of the present invention to provideimmunostimulating agents, which act as adjuvants and stimulate theinnate immune system, preferably if administered in combination withother biologically active compounds, in particular if administeredtogether with immune-modulating compounds, more preferably incombination with compounds, which specifically stimulate the adaptiveimmune system, such as antigens.

In this context, it is known that (unspecific) immunostimulating effectscan also be produced by directly using nucleic acids to trigger anunspecific (i.e. innate) immune response, e.g. with bacterial CpG-DNAsequences, which not only serve for genetic information. For example,DNA is known to play a central role in the production of unspecificimmune responses. Bacterial DNA, for example, is known to act as“danger” signal to alert immune cells, such as macrophages and dendriticcells and to promote protective Th1 polarized T cell immune responses.An immunostimulating action appears to result from the presence ofunmethylated CG (nucleic acid) motifs, and such CpG-DNA has thereforebeen proposed as an immunostimulating agent as such (see e.g. U.S. Pat.No. 5,663,153). CpG-DNA directly causes activation of members of theinnate immune system yielding in up-regulation of co-stimulatorymolecules and pro-inflammatory cytokines. This immunostimulatingproperty of DNA can also be achieved by DNA oligonucleotides which arestabilized by phosphorothioate modification (see e.g. U.S. Pat. No.6,239,116). Such immunostimulating DNA may also be combined with furtherimmunostimulating compounds. E.g., U.S. Pat. No. 6,406,705 disclosesimmunostimulating compositions which contain a synergistic combinationof a CpG oligodeoxyribonucleotide and a non-nucleic acid compound toexert a stimulating effect on the innate immune system.

However, the use of DNA to exert an unspecific immune response can beless advantageous from several points of view. DNA is decomposed onlyrelatively slowly in vivo so that, when immunostimulating (foreign) DNAis used, the formation of anti-DNA antibodies may occur, which has beenconfirmed in an animal model in mouse (Gilkeson et al., J. Clin. Invest.1995, 95: 1398-1402). Persistence of (foreign) DNA in the organism canthus lead to over-activation of the immune system, which is known inmice to result in splenomegaly (Montheith et al., Anticancer Drug Res.1997, 12(5): 421-432). Furthermore, (foreign) DNA can interact with thehost genome and cause mutations, in particular by integration into thehost genome. For example, insertion of the introduced (foreign) DNA intoan intact gene can occur, which represents a mutation which can impedeor even eliminate completely the function of the endogenous gene. As aresult of such integration events enzyme systems that are vital to thecell can be destroyed. However, there is also a risk that the cell sochanged will be transformed into a degenerate state. Such transformationmay occur e.g. if, by the integration of the (foreign) DNA, a gene thatis critical for the regulation of cell growth is changed. Therefore, inprocesses known hitherto, a possible risk of cancer formation cannot beruled out when using (foreign) DNA as immunostimulating agent.

It is therefore generally more advantageous to use specific RNAmolecules as a compound to elicit an (unspecific) response of the innateimmune system. In this context, the innate immune system as part of theimmune system is the dominant system of host defense in most organismsand comprises barriers such as humoral and chemical barriers including,e.g., inflammation, the complement system and cellular barriers.Additionally, the innate immune system is based on a small number ofreceptors, called pattern recognition receptors or pathogen associatedmolecular pattern receptors (PAMP-receptors), such as members of theToll-like receptor (TLR) family (see e.g. Trinchieri and Sher, Naturereviews, Immunology, Volume 7, March 2007). Such TLRs are transmembraneproteins which recognize ligands of the extracellular milieu or of thelumen of endosomes. Following ligand-binding they transduce the signalvia cytoplasmic adaptor proteins which leads to triggering of ahost-defence response and entailing production of antimicrobialpeptides, proinflammatory chemokines and cytokines, antiviral cytokines,etc. (see e.g. Meylan, E., J. Tschopp, et al. (2006). “Intracellularpattern recognition receptors in the host response.” Nature 442(7098):39-44).

To date, at least 10 members of Toll-like receptors (TLRs) have beenidentified in human and 13 in mice, which are in part identified withrespect to their mode of action. In humans, those Toll-like receptors(TLRs) include TLR1-TLR2 (known ligand: Triacyl lipopeptide), TLR1-TLR6(known ligand: Diacyl lipopeptide), TLR2 (known ligand: Peptidoglycan),TLR3 (known ligand: dsRNA), TLR4 (known ligand: LPS (lipopolysachharide)of Gram-negative bacteria)), TLR5 (known ligand: bacterialflagellin(s)), TLR7/8 (known ligands: imidazoquinolines, guanosine(guanine) analogs and ssRNA), TLR9 (known ligands: CpG DNA of bacteria,viruses and protozoans and malaria pigment hemozoin (product ofdigestion of haemoglobin)) and TLR10. After recognition of microbialpathogens, these TLRs typically trigger intracellular signallingpathways that result in induction of inflammatory cytokines (e.g.TNF-alpha, IL-6, IL-1-beta and IL-12), type I interferon (IFN-beta andmultiple IFN-alpha) and chemokines (Kawai, T. and S. Akira (2006). “TLRsignaling.” Cell Death Differ 13(5): 816-25).

In this context, RNAs are advantageous for several reasons. E.g., asknown today and mentioned above, ssRNA is capable of binding to TLR-7/-8receptors and dsRNA is capable of binding to TLR receptors and therebyexerting an immunostimulating effect. Furthermore, RNA asimmunostimulating agent typically has a substantially shorter half-lifein vivo than DNA, thereby avoiding the above mentioned drawbacks of DNA.Nevertheless, the use of those specific RNA molecules known asimmunostimulating agents in the art also has some limitations. Forexample, the specific RNA sequences disclosed hitherto in the artexhibit only limited immunostimulating capacities in vivo. This mayrequire an increased amount of RNA for immunostimulation, which,regardless of the increased costs owing to the increased amounts of RNAto be administered, involves the risk of the mostly undesirableside-effects described generally hereinbefore, for example irritationand inflammation at the site of administration, even if this may be thecase for a limited time window. Also, toxic side-effects cannot be ruledout when large amounts of the immunostimulating agent are administered.

A further limitation is the low induction of type I interferons (e.g.IFNalpha and IFNbeta) by known immunostimulating RNA molecules which areimportant inducers of antiviral and antiproliferative activities andcytolytic activity in lymphocytes, natural killer cells and macrophages.

Known immunostimulating dsRNA molecules are for instance poly A:U andpoly I:C. The disadvantage of these immunostimulating dsRNA molecules,however, is their undefined length, which may lead to non-predictablemolecular structures and thereby to aggregates. Such aggregates mayfurther lead to undesired side effects such as occlusion of bloodvessels or undue immunostimulation at the site of injection.Additionally, such non-predictable molecular structures represent aproblem in daily laboratory and production routines as no adequatequality control may be carried out due to variable product parameters.Here, a defined nucleic acid molecule exhibiting a defined length andstructure and being suitable as an adjuvant is preferred forpharmaceutical applications.

Despite the success of RNA demonstrated hitherto, there is therefore acontinued need for, and considerable interest in, improvedimmunostimulating agents which may exert by their own an immune responseof the patient's innate immune system. Accordingly, it is a secondobject of the invention to provide immunostimulating agents which exertan unspecific immune response by activating the patient's innate immunesystem.

Both objects of the present invention are solved by the provision ofnucleic acid molecules of the following generic formula (I). Theseinventive nucleic acid molecules activate the innate immune system, thuseliciting an unspecific immune response. As adjuvants (e.g. as componentof a vaccine), they may additionally support the immunostimulatingactivity of a second compound specifically activating the adaptiveimmune system.

The present invention provides a nucleic acid (molecule) of formula (I):

(N_(u)G_(k)X_(m)G_(n)N_(v))_(a),

wherein:

-   G is guanosine (guanine), uridine (uracil) or an analogue of    guanosine (guanine) or uridine (uracil), preferably guanosine    (guanine) or an analogue thereof;-   X is guanosine (guanine), uridine (uracil), adenosine (adenine),    thymidine (thymine), cytidine (cytosine), or an analogue of these    nucleotides (nucleosides), preferably uridine (uracil) or an    analogue thereof;-   N is a nucleic acid sequence having a length of about 4 to 50,    preferably of about 4 to 40, more preferably of about 4 to 30 or 4    to 20 nucleic acids, each N independently being selected from    guanosine (guanine), uridine (uracil), adenosine (adenine),    thymidine (thymine), cytidine (cytosine) or an analogue of these    nucleotides (nucleosides);-   a is an integer from 1 to 20, preferably from 1 to 15, most    preferably from 1 to 10;-   l is an integer from 1 to 40,    -   wherein when l=1, G is guanosine (guanine) or an analogue        thereof,        -   when l>1, at least 50% of these nucleotides (nucleosides)            are guanosine (guanine) or an analogue        -   thereof;-   m is an integer and is at least 3;    -   wherein when m=3, X is uridine (uracil) or an analogue thereof,        and        -   when m>3, at least 3 successive uridines (uracils) or            analogues of uridine (uracil) occur;-   n is an integer from 1 to 40,    -   wherein when n=1, G is guanosine (guanine) or an analogue        thereof,        -   when n>1, at least 50% of these nucleotides (nucleosides)            are guanosine (guanine) or an analogue        -   thereof;-   u,v may be independently from each other an integer from 0 to 50,    -   preferably wherein when u=0, v≧1, or        -   when v=0, u≧1;            wherein the nucleic acid molecule of formula (I) according            to the invention has a length of at least 50 nucleotides,            preferably of at least 100 nucleotides, more preferably of            at least 150 nucleotides, even more preferably of at least            200 nucleotides and most preferably of at least 250            nucleotides.

The molecule (N_(u)G_(l)X_(m)G_(n)N_(v))_(a) of formula (I) according tothe invention is typically a nucleic acid, which may be in the form ofany DNA or RNA, preferably, without being limited thereto, a circular orlinear DNA or RNA, a single- or a double-stranded DNA or RNA (which mayalso be regarded as an DNA or RNA due to non-covalent association of twosingle-stranded DNAs or RNAs) or a partially double-stranded DNA or RNA(which is typically formed by a longer and at least one shortersingle-stranded DNA or RNA molecule or by at least two single-strandedDNA or RNA-molecules, which are about equal in length, wherein one ormore single-stranded DNA or RNA molecules are in part complementary toone or more other single-stranded DNA or RNA molecules and thus form adouble-stranded RNA in this region), e.g. a (partially) single-strandedDNA or RNA, mixed with regions of a (partially) double-stranded DNA orRNA. Preferably, the nucleic acid molecule of formula (I) according tothe invention may be in the form of a single- or a double-stranded DNAor RNA, more preferably a partially double-stranded DNA or RNA. It isalso preferred that the nucleic acid molecule of formula (I) accordingto the invention is in the form of a mixture of a single-strandednucleic and double stranded DNA or RNA.

It is particularly advantageous, if the inventive nucleic acid(N_(u)G_(l)X_(m)G_(n)N_(v))_(a) of formula (I) according to theinvention is a partially double-stranded nucleic acid molecule, sincesuch a (partially double-stranded) inventive nucleic acid moleculeaccording to formula (I) (or of formula (Ia), (II) (IIa), (IIb), (IIIc)and/or (IIIb) as defined below), can positively stimulate the innateimmune response in a patient to be treated by addressing thePAMP—(pathogen associated molecular pattern) receptors forsingle-stranded RNA (TLR-7 and TLR-8) as well as the PAMP-receptors fordouble-stranded RNA (TLR-3, RIG-I and MDA-5). Receptors TLR-3, TLR-7 andTLR-8 are located in the endosome and are activated by RNA taken up bythe endosome. In contrast, RIG-I and MDA-5 are cytoplasmic receptors,which are activated by RNA, which was directly taken up into thecytoplasm or which has been released from the endosomes (endosomalrelease or endosomal escape). Accordingly, any partially double-strandedinventive nucleic acid (N_(u)G_(l)X_(m)G_(n)N_(v))_(a) of formula (I)(or (a partially double-stranded) inventive nucleic acid moleculeaccording to formula (I) (and (Ia), (II) (IIa), (IIb), (IIIc) and (IIIb)as defined below)) is capable of activating different signal cascades ofimmunostimulation and thus leads to an innate immune response orenhances such a response significantly.

The structure (N_(u)G_(l)X_(m)G_(n)N_(v))_(a) of formula (I) accordingto the present invention comprises the element G_(l)X_(m)G_(n) as a corestructure and additionally the bordering elements N_(u) and/or N_(v),wherein the whole element N_(u)G_(l)X_(m)G_(n)N_(v) may occurrepeatedly, i.e. at least once, as determined by the integer a. In thiscontext, the inventors surprisingly found, that a molecule according toformula (I) according to the invention, i.e. having the structure(N_(u)G_(l)X_(m)G_(n)N_(v))_(a) as defined above, leads to an increasedinnate immune response in a patient, which is particularly indicated byan increase of IFNalpha release, when compared to administration of thecore structure G_(l)X_(m)G_(n) as such. Furthermore, a moleculecomprising the above core structure G_(l)X_(m)G_(n) can be amplified inbacterial organisms with a significantly better yield, when it isbordered by a repetitive element N_(u) and/or N_(v) as defined informula (I). This molecule design is particularly advantageous whenpreparing a molecule according structure (N_(u)G_(l)X_(m)G_(n)N_(v))_(a)of formula (I) as defined above by using in vitro transcription methodsinstead of solid phase synthesis methods as known in the art, which aretypically limited to a specific size of nucleic acids.

The core structure G_(l)X_(m)G_(n) of formula (I) according to theinvention is defined more closely in the following:

G in the nucleic acid molecule of formula (I) according to the inventionis a nucleotide or deoxynucleotide or comprises a nucleoside, whereinthe nucleotide (nucleoside) is guanosine (guanine) or uridine (uracil)or an analogue thereof, more preferably guanosine (guanine) or ananalogue thereof. In this connection, guanosine (guanine) or uridine(uracil) nucleotide (nucleoside) analogues are defined as non-nativelyoccurring variants of the naturally occurring nucleotides (nucleoside)guanosine (guanine) and uridine (uracil). Accordingly, guanosine(guanine) or uridine (uracil) analogues are typically chemicallyderivatized nucleotides (nucleoside) with non-natively occurringfunctional groups or components, which are preferably added to, modifiedor deleted from the naturally occurring guanosine (guanine) or uridine(uracil) nucleotide or which substitute the naturally occurringfunctional groups or components of a naturally occurring guanosine(guanine) or uridine (uracil) nucleotide. Accordingly, each functionalgroup or component of the naturally occurring guanosine (guanine) oruridine (uracil) nucleotide may be modified or deleted therefrom, namelythe base component, the sugar (ribose) component, any naturallyoccurring functional side group and/or the phosphate component formingthe oligonucleotide's backbone. The phosphate moieties may besubstituted by e.g. phosphoramidates, phosphorothioates, peptidenucleotides, methylphosphonates etc., however, naturally occurringphosphodiester backbones still being preferred in the context of thepresent invention. Additionally, the sugar (ribose) component isselected from a desoxyribose, particularly the nucleic acid is an RNA asdefined above, wherein the sugar (ribose) component is selected from adesoxyribose.

Accordingly, analogues of guanosine (guanine) or uridine (uracil)include, without implying any limitation, any naturally occurring ornon-naturally occurring guanosine (guanine) or uridine (uracil) that hasbeen altered chemically, for example by acetylation, methylation,hydroxylation, etc., including, for example, 1-methyl-guanosine(guanine), 2-methyl-guanosine (guanine), 2,2-dimethyl-guanosine(guanine), 7-methyl-guanosine (guanine), dihydro-uridine (uracil),4-thio-uridine (uracil), 5-carboxymethylaminomethyl-2-thio-uridine(uracil), 5-(carboxy-hydroxylmethyl)-uridine (uracil), 5-fluoro-uridine(uracil), 5-bromo-uridine (uracil), 5-carboxymethylaminomethyl-uridine(uracil), 5-methyl-2-thio-uridine (uracil), N-uridine(uracil)-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uridine(uracil), 5-methoxyaminomethyl-2-thio-uridine (uracil),5′-methoxycarbonylmethyl-uridine (uracil), 5-methoxy-uridine (uracil),uridine (uracil)-5-oxyacetic acid methyl ester, uridine(uracil)-5-oxyacetic acid (v). The preparation of such analogues isknown to a person skilled in the art, for example from U.S. Pat. No.4,373,071, U.S. Pat. No. 4,401,796, U.S. Pat. No. 4,415,732, U.S. Pat.No. 4,458,066, U.S. Pat. No. 4,500,707, U.S. Pat. No. 4,668,777, U.S.Pat. No. 4,973,679, U.S. Pat. No. 5,047,524, U.S. Pat. No. 5,132,418,U.S. Pat. No. 5,153,319, U.S. Pat. No. 5,262,530 and U.S. Pat. No.5,700,642, the disclosures of which are incorporated by reference hereinin their entirety. In the case of an analogue as described above,preference is given according to the invention especially to thoseanalogues that increase the immunogenity of the nucleic acid molecule offormula (I) according to the invention and/or do not interfere with afurther modification that has been introduced. At least one guanosine(guanine) or uridine (uracil) or an analogue thereof can occur in thecore structure elements G_(l) and/or G_(n), optionally at least 10%,20%, 30%, 40%, 50%, 60%, 70%, 80% 90% or even 100% of the nucleotides ofthe core structure elements G_(l) and/or G_(n) are a naturally occurringguanosine (guanine), a naturally occurring uridine (uracil), and/or ananalogue thereof and/or exhibit properties of an analogue thereof asdefined herein. Preferably, the core structure element G_(l) and/orG_(n) contains at least one analogue of a naturally occurring guanosine(guanine) and/or a naturally occurring uridine (uracil) at all. Mostpreferably, all nucleotides (nucleosides) of these core structureelements G_(l) and/or G_(n) are analogues, which may—most preferably—beidentical analogues for the same type of nucleotides (nucleosides) (e.g.all guanosine (guanine) nucleotides are provided as 1-methyl-guanosine(guanine)) or they may be distinct (e.g. at least two different guanosinanalogues substitute the naturally occurring guanosin nucleotide).

The number of nucleotides (nucleosides) of core structure element G(G_(l) and/or G_(n)) in the nucleic acid molecule of formula (I)according to the invention is determined by l and n. l and n,independently of one another, are each an integer from 1 to 100, 1 to90, 1 to 80, 1 to 70, 1 to 60, preferably 1 to 50, yet more preferably 1to 40, and even more preferably 1 to 30, wherein the lower limit ofthese ranges may be 1, but alternatively also 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, or even more. Preferably, for each integer, when l and/orn=1, G is guanosine (guanine) or an analogue thereof, and when l or n>1,at least 50%, more preferably at least 50%, 60%, 70%, 80%, 90% or even100% of the nucleotides (nucleosides) of core structure element G (G_(l)and/or G_(n)) are guanosine (guanine) or an analogue thereof. Forexample, without implying any limitation, when l or n=4, G_(l) and/orG_(n) can be, for example, a GUGU, GGUU, UGUG, UUGG, GUUG, GGGU, GGUG,GUGG, UGGG or GGGG, etc.; when l or n=5, G_(l) and/or G_(n) can be, forexample, a GGGUU, GGUGU, GUGGU, UGGGU, UGGUG, UGUGG, UUGGG, GUGUG,GGGGU, GGGUG, GGUGG, GUGGG, UGGGG, or GGGGG, etc.; etc. A nucleotide(nucleoside) of core structure elements G_(l) and/or G_(n) directlyadjacent to X_(m) in the nucleic acid molecule of formula (I) accordingto the invention is preferably not an uridine (uracil) or an analoguethereof. More preferably nucleotides (nucleosides) of core structureelements G_(l) and/or G_(n) directly adjacent to X_(m) in the nucleicacid molecule of formula (I) according to the invention are at least oneguanosine (guanine) or an analogue thereof, more preferably a stretch ofat least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19or even 20 or more guanosines (guanines) or an analogue thereof.Additionally, a nucleotide of core structure elements G_(l) and/or G_(n)directly adjacent to N, e.g. N_(u), and/or N_(v) (or N_(w1) or N_(w2) asdefined below) in the nucleic acid molecule of formula (I) according tothe invention is preferably not an uridine (uracil) or an analoguethereof. More preferably, nucleotides (nucleosides) of core structureelements G_(l) and/or G_(n) directly adjacent to N, e.g. N_(u), and/orN_(v) (or N_(w1) or N_(w2) as defined below) in the nucleic acidmolecule of formula (I) according to the invention are at least oneguanosine (guanine) or an analogue thereof, more preferably a stretch ofat least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19or even 20 or more guanosines (guanines) or an analogue thereof.

The term “identity” in the present application means that the sequencesare compared in relation to a reference sequence and the percentageidentity is determined by comparing them. For example, in order todetermine the percentage identity of two nucleic acid sequences, thesequences can first be arranged relative to one another (alignment) inorder to permit subsequent comparison of the sequences. To this end, forexample, gaps can be introduced into the sequence of the first nucleicacid sequence and the nucleotides can be compared with the correspondingposition of the second nucleic acid sequence. When a position in thefirst nucleic acid sequence is occupied with the same nucleotide as in aposition in the second sequence, then the two sequences are identical atthat position. The percentage identity between two sequences is afunction of the number of identical positions divided by the sequences.If, for example, a specific sequence identity is assumed for aparticular nucleic acid in comparison with a reference nucleic acidhaving a defined length, then this percentage identity is indicatedrelatively in relation to the reference nucleic acid. Therefore,starting, for example, from a nucleic acid sequence that has 50%sequence identity with a reference nucleic acid sequence having a lengthof 100 nucleotides, that nucleic acid sequence can represent a nucleicacid sequence having a length of 50 nucleotides that is wholly identicalwith a section of the reference nucleic acid sequence having a length of50 nucleotides. It can, however, also represent a nucleic acid sequencehaving a length of 100 nucleotides that has 50% identity, that is to sayin this case 50% identical nucleic acids, with the reference nucleicacid sequence over its entire length. Alternatively, that nucleic acidsequence can be a nucleic acid sequence having a length of 200nucleotides that, in a section of the nucleic acid sequence having alength of 100 nucleotides, is wholly identical with the referencenucleic acid sequence having a length of 100 nucleotides. Other nucleicacid sequences naturally fulfil these criteria equally.

The determination of the percentage identity of two sequences can becarried out by means of a mathematical algorithm. A preferred butnon-limiting example of a mathematical algorithm which can be used forcomparing two sequences is the algorithm of Karlin et al. (1993), PNASUSA, 90:5873-5877. Such an algorithm is integrated into the NBLASTprogram, with which sequences having a desired identity with thesequences of the present invention can be identified. In order to obtaina gapped alignment as described above, the “Gapped BLAST” program can beused, as described in Altschul et al. (1997), Nucleic Acids Res,25:3389-3402. When using BLAST and Gapped BLAST programs, the defaultparameters of the particular program (e.g. NBLAST) can be used. Thesequences can further be aligned using version 9 of GAP (globalalignment program) from “Genetic Computing Group”, using the default(BLOSUM62) matrix (values −4 to +11) with a gap open penalty of −12 (forthe first zero of a gap) and a gap extension penalty of −4 (for eachadditional successive zero in the gap). After the alignment, thepercentage identity is calculated by expressing the number ofcorrespondences as a percentage of the nucleic acids in the claimedsequence. The described methods for determining the percentage identityof two nucleic acid sequences can also be applied correspondingly toamino acid sequences using the appropriate programs.

Likewise preferably, for formula (I), when l or n>1, at least 60%, 70%,80%, 90% or even 100% of the nucleotides (nucleosides) of the corestructure elements G_(l) and/or G_(n) are guanosine (guanine) or ananalogue thereof, as defined above. The remaining nucleotides(nucleosides) to 100% in the core structure elements G_(l) and/or G_(n)(when guanosine (guanine) constitutes less than 100% of thesenucleotides (nucleosides)) may then be uridine (uracil) or an analoguethereof, as defined hereinbefore.

X, particularly X_(m), in the nucleic acid molecule of formula (I)according to the invention is also a core structure element and is anucleotide or deoxynucleotide or comprises a nucleoside, wherein thenucleotide (nucleoside) is typically selected from guanosine (guanine),uridine (uracil), adenosine (adenine), thymidine (thymine), cytidine(cytosine) or an analogue thereof, preferably uridine (uracil) or ananalogue thereof. In this connection, nucleotide (nucleoside) analoguesare defined as non-natively occurring variants of naturally occurringnucleotides (nucleosides). Accordingly, analogues are chemicallyderivatized nucleotides (nucleosides) with non-natively occurringfunctional groups, which are preferably added to or deleted from thenaturally occurring nucleotide (nucleoside) or which substitute thenaturally occurring functional groups of a nucleotide (nucleoside).Accordingly, each component of the naturally occurring nucleotide may bemodified, namely the base component, the sugar (ribose or desoxyribose)component and/or the phosphate component forming the oligonucleotide'sbackbone. The phosphate moieties may be substituted by e.g.phosphoramidates, phosphorothioates, peptide nucleotides,methylphosphonates etc., wherein, however, the naturally occurringphosphodiester backbone is still preferred. Preferably, at least 10%,more preferably at least 20%, more preferably at least 30%, morepreferably at least 50%, more preferably at least 70% and even morepreferably at least 90% of all “X” nucleotides may exhibit properties ofan analogue as defined herein, if the inventive nucleic acid contains atleast one analogue at all. The analogues substituting a specificnucleotide type within the core structure element “X_(m)” may beidentical, e.g. all cytidine (cytosine) nucleotides (nucleosides)occurring in the core structure element “X_(m)” are formed by a specificcytidine (cytosine) analogue, e.g. 2-thio-cytidine (cytosine), or theymay be distinct for a specific nucleotide (nucleosides), e.g. at leasttwo distinct cytidine (cytosine) analogues are contained within the corestructure element “X_(m)”.

Analogues of guanosine (guanine), uridine (uracil), adenosine (adenine),thymidine (thymine), cytidine (cytosine) include, without implying anylimitation, any naturally occurring or non-naturally occurring guanosine(guanine), uridine (uracil), adenosine (adenine), thymidine (thymine) orcytidine (cytosine) that has been altered chemically, for example byacetylation, methylation, hydroxylation, etc., including1-methyl-adenosine (adenine), 2-methyl-adenosine (adenine),2-methylthio-N6-isopentenyl-adenosine (adenine), N6-methyl-adenosine(adenine), N6-isopentenyl-adenosine (adenine), 2-thio-cytidine(cytosine), 3-methyl-cytidine (cytosine), 4-acetyl-cytidine (cytosine),2,6-diaminopurine, 1-methyl-guanosine (guanine), 2-methyl-guanosine(guanine), 2,2-dimethyl-guanosine (guanine), 7-methyl-guanosine(guanine), inosine, 1-methyl-inosine, dihydro-uridine (uracil),4-thio-uridine (uracil), 5-carboxymethylaminomethyl-2-thio-uridine(uracil), 5-(carboxyhydroxylmethyl)-uridine (uracil), 5-fluoro-uridine(uracil), 5-bromo-uridine (uracil), 5-carboxymethylaminomethyl-uridine(uracil), 5-methyl-2-thio-uridine (uracil), N-uridine(uracil)-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uridine(uracil), 5-methoxyaminomethyl-2-thio-uridine (uracil),5′-methoxycarbonylmethyl-uridine (uracil), 5-methoxy-uridine (uracil),uridine (uracil)-5-oxyacetic acid methyl ester, uridine(uracil)-5-oxyacetic acid (v), queosine, beta-D-mannosyl-queosine,wybutoxosine, and inosine. The preparation of such analogues is known toa person skilled in the art, for example from U.S. Pat. No. 4,373,071,U.S. Pat. No. 4,401,796, U.S. Pat. No. 4,415,732, U.S. Pat. No.4,458,066, U.S. Pat. No. 4,500,707, U.S. Pat. No. 4,668,777, U.S. Pat.No. 4,973,679, U.S. Pat. No. 5,047,524, U.S. Pat. No. 5,132,418, U.S.Pat. No. 5,153,319, U.S. Pat. No. 5,262,530 and U.S. Pat. No. 5,700,642.In the case of an analogue as described above, particular preference isgiven according to the invention to those analogues of nucleotides(nucleosides) that increase the immunogenity of the nucleic acidmolecule of formula (I) according to the invention and/or do notinterfere with a further modification that has been introduced.

The number of core structure element X in the nucleic acid molecule offormula (I) according to the invention is determined by m. m is aninteger and is typically at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60to 70, 70 to 80, 80 to 90, 90 to 100, 100 to 150, 150 to 200, or evenmore, wherein when m=3, X is uridine (uracil) or an analogue thereof,and when m>3, at least 3 or more directly successive uridines (uracils)or an analogue thereof occur in the element X of formula (I) above. Sucha sequence of at least 3 or more directly successive uridines (uracils)is referred to in connection with this application as a “monotonicuridine (uracil) sequence”. A monotonic uridine (uracil) sequencetypically has a length of at least 3, 4, 5, 6, 7, 8, 9 or 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60,60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to 150, 150 to 200 uridines(uracils) or optionally analogues of uridine (uracil) as defined above.Such a monotonic uridine (uracil) sequence occurs at least once in thecore structure element X of the nucleic acid molecule of formula (I)according to the invention. It is therefore possible, for example, for1, 2, 3, 4, 5 or more monotonic uridine (uracil) sequences having atleast 3 or more uridines (uracils) or analogues thereof to occur, whichmonotonic uridine (uracil) sequences can be interrupted in the corestructure element X by at least one guanosine (guanine), adenosine(adenine), thymidine (thymine), cytidine (cytosine) or an analoguethereof, preferably 2, 3, 4, 5 or more. For example, when m=3, X_(m) isa UUU. When m=4, X_(m) can be, for example, without implying anylimitation, a UUUA, UUUG, UUUC, UUUU, AUUU, GUUU or CUUU, etc. Whenn=10, X_(m) can be, for example, without implying any limitation, aUUUAAUUUUC (SEQ ID NO: 120), UUUUGUUUUA (SEQ ID NO: 121), UUUGUUUGUU(SEQ ID NO: 122), UUGUUUUGUU (SEQ ID NO: 123), UUUUUUUUUU (SEQ ID NO:124), etc. The nucleotides of X_(m) adjacent to G_(l) or G_(n) of thenucleic acid molecule of formula (I) according to the inventionpreferably comprise uridine (uracil) or analogues thereof. When m>3,typically at least 50%, preferably at least 60%, 70%, 80%, 90% or even100%, of the nucleotides of X_(m) are uridine (uracil) or an analoguethereof, as defined above. The remaining nucleotides of X_(m) to 100%(where there is less than 100% uridine (uracil) in the sequence X_(m))are then guanosine (guanine), uridine (uracil), adenosine (adenine),thymidine (thymine), cytidine (cytosine) or an analogue thereof, asdefined above.

The inventive nucleic acid according formula (I) above also containsbordering element N. The bordering element N is typically a nucleic acidsequence having a length of about 4 to 50, preferably of about 4 to 40,more preferably of about 4 to 30 nucleotides (nucleosides), even morepreferably of about 4 to 20 nucleotides (nucleosides), wherein the lowerlimit of these ranges alternatively also may be 5, 6, 7, 8, 9, 10, ormore. Preferably, the nucleotides (nucleosides) of each N areindependently selected from guanosine (guanine), uridine (uracil),adenosine (adenine), thymidine (thymine), cytidine (cytosine) and/or ananalogue thereof. In other words, bordering element N in the nucleicacid molecule of formula (I) according to the present invention may be asequence, which may be composed of any (random) sequence, available inthe art, each N independently selected from guanosine (guanine), uridine(uracil), adenosine (adenine), thymidine (thymine), cytidine (cytosine)and/or an analogue of these nucleotides, or from a homopolymer of thesenucleotides (nucleosides), in each case provided, that such a sequencehas a length of about 4 to 50, preferably of about 4 to 40, morepreferably of about 4 to 30 nucleotides (nucleosides) and even morepreferably of about 4 to 30 or 4 to 20 nucleotides (nucleosides)according to the above definition.

According to a specific embodiment, N may be a nucleic acid sequencewithin the above definitions, wherein the sequence typically comprisesnot more than 2 identical nucleotides (nucleosides) as defined above ina directly neighboring position, i.e. the sequence typically comprisesno stretches of more than two identical nucleotides (nucleosides)selected from adenosine (adenine), cytidine (cytosine), uridine (uracil)and/or guanosine (guanine), and/or an analogue thereof (i.e. a stretchof “aa”, “cc”, “uu”, “gg” and/or an analogue thereof), more preferablyno such stretch, i.e. no identical nucleotides (nucleosides) as definedabove in a directly neighboring position. Additionally or alternatively,N may be a nucleic acid sequence within the above definitions, whereinthe sequence typically comprises a content of adenosine (adenine) or ananalogue thereof preferably of about 0 to 50%, 5 to 45%, or 10 to 40%,more preferably of about 15 to 35%, even more preferably of about 20 to30%, and most preferably of about 25%; a content of uridine (uracil) oran analogue thereof preferably of about 0 to 50%, 5 to 45%, or 10 to40%, more preferably of about 15 to 35%, even more preferably of about20 to 30%, and most preferably of about 25%; a content of cytidine(cytosine) or an analogue thereof preferably of about 0 to 50%, 5 to45%, or 10 to 40%, more preferably of about 15 to 35%, even morepreferably of about 20 to 30%, and most preferably of about 25%; acontent of guanosine (guanine) or an analogue thereof preferably ofabout 0 to 50%, 5 to 45%, or 10 to 40%, more preferably of about 15 to35%, even more preferably of about 20 to 30%, and most preferably ofabout 25%. Most preferably, N may be a nucleic acid sequence within theabove definitions, wherein the sequence typically comprises a content ofeach adenosine (adenine), guanosine (guanine), cytidine (cytosine) anduridine (uracil) of about 25%. Examples of such sequences of N includee.g. agcu, aguc, augc, acgu, gcua, gcau, gacu, guca, cuag, caug, cagu,cgau, uagc, uacg, ucga, ucag, agcugcua, gcaucaug, caguucga, etc.,

The number of bordering element N in the nucleic acid molecule offormula (I) according to the invention, i.e. its repetition, isdetermined by integers u and/or v. Thus, N in the nucleic acid moleculeof formula (I) according to the invention may occur as a (repetitive)bordering element N_(u) and/or N_(v), wherein u and/or v may be,independently from each other, an integer from 0 or 1 to 100, morepreferably from 0 or 1 to 50, even more preferably from 0 or 1 to 40,and most preferably from 0 or 1 to 30, e.g. 0 or 1 to 5, 10, 20, 25, or30; or from 5 to 10, 10 to 15, 15 to 20, 20 to 25 or 25 to 30. Morepreferably, at least one (repetitive) bordering element N_(u) and/orN_(v), may be present in formula (I), i.e. either u or v are not 0, morepreferably, both (repetitive) bordering elements N_(u) and/or N_(v) arepresent, even more preferably in the above definitions.

Additionally, the combination of core structure elements and borderingelements to the element N_(u)G_(l)X_(m)G_(n)N_(v) may occur asrepetitive elements according to the inventive molecule of formula (I),(N_(u)G_(l)X_(m)G_(n)N_(v))_(a), as defined above, wherein the number ofrepetitions of the combined element according to formula (I),(N_(u)G_(l)X_(m)G_(n)N_(v))_(a), is determined by the integer a.Preferably, a is an integer from about 1 to 100, 1 to 50, 1 to 20, morepreferably an integer from about 1 to 15, most preferably an integerfrom about 1 to 10. In this context, the repetitive elementsN_(u)G_(l)X_(m)G_(n)N_(v) may be equal or different from each other.

According to a particularly preferred embodiment, the inventive nucleicacid molecule of formula (I) (N_(u)G_(l)X_(m)G_(n)N_(v))_(a), as definedabove, comprises a core structure G_(l)X_(m)G_(n), preferably selectedfrom at least one of the following sequences of SEQ ID NOs: 1-80:

(SEQ ID NO: 1) GGUUUUUUUUUUUUUUUGGG; (SEQ ID NO: 2)GGGGGUUUUUUUUUUGGGGG; (SEQ ID NO: 3)GGGGGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGGGGG; (SEQ ID NO: 4)GUGUGUGUGUGUUUUUUUUUUUUUUUUGUGUGUGUGUGU; (SEQ ID NO: 5)GGUUGGUUGGUUUUUUUUUUUUUUUUUGGUUGGUUGGUU; (SEQ ID NO: 6)GGGGGGGGGUUUGGGGGGGG; (SEQ ID NO: 7) GGGGGGGGUUUUGGGGGGGG;(SEQ ID NO: 8) GGGGGGGUUUUUUGGGGGGG; (SEQ ID NO: 9)GGGGGGGUUUUUUUGGGGGG; (SEQ ID NO: 10) GGGGGGUUUUUUUUGGGGGG;(SEQ ID NO: 11) GGGGGGUUUUUUUUUGGGGG; (SEQ ID NO: 12)GGGGGGUUUUUUUUUUGGGG; (SEQ ID NO: 13) GGGGGUUUUUUUUUUUGGGG;(SEQ ID NO: 14) GGGGGUUUUUUUUUUUUGGG; (SEQ ID NO: 15)GGGGUUUUUUUUUUUUUGGG; (SEQ ID NO: 16) GGGGUUUUUUUUUUUUUUGG;(SEQ ID NO: 17) GGUUUUUUUUUUUUUUUUGG; (SEQ ID NO: 18)GUUUUUUUUUUUUUUUUUUG; (SEQ ID NO: 19) GGGGGGGGGGUUUGGGGGGGGG;(SEQ ID NO: 20) GGGGGGGGGUUUUGGGGGGGGG; (SEQ ID NO: 21)GGGGGGGGUUUUUUGGGGGGGG; (SEQ ID NO: 22) GGGGGGGGUUUUUUUGGGGGGG;(SEQ ID NO: 23) GGGGGGGUUUUUUUUGGGGGGG; (SEQ ID NO: 24)GGGGGGGUUUUUUUUUGGGGGG; (SEQ ID NO: 25) GGGGGGGUUUUUUUUUUGGGGG;(SEQ ID NO: 26) GGGGGGUUUUUUUUUUUGGGGG; (SEQ ID NO: 27)GGGGGGUUUUUUUUUUUUGGGG; (SEQ ID NO: 28) GGGGGUUUUUUUUUUUUUGGGG;(SEQ ID NO: 29) GGGGGUUUUUUUUUUUUUUGGG; (SEQ ID NO: 30)GGGUUUUUUUUUUUUUUUUGGG; (SEQ ID NO: 31) GGUUUUUUUUUUUUUUUUUUGG;(SEQ ID NO: 32) GGGGGGGGGGGUUUGGGGGGGGGG; (SEQ ID NO: 33)GGGGGGGGGGUUUUGGGGGGGGGG; (SEQ ID NO: 34) GGGGGGGGGUUUUUUGGGGGGGGG;(SEQ ID NO: 35) GGGGGGGGGUUUUUUUGGGGGGGG; (SEQ ID NO: 36)GGGGGGGGUUUUUUUUGGGGGGGG; (SEQ ID NO: 37) GGGGGGGGUUUUUUUUUGGGGGGG;(SEQ ID NO: 38) GGGGGGGGUUUUUUUUUUGGGGGG; (SEQ ID NO: 39)GGGGGGGUUUUUUUUUUUGGGGGG; (SEQ ID NO: 40) GGGGGGGUUUUUUUUUUUUGGGGG;(SEQ ID NO: 41) GGGGGGUUUUUUUUUUUUUGGGGG; (SEQ ID NO: 42)GGGGGGUUUUUUUUUUUUUUGGGG; (SEQ ID NO: 43) GGGGUUUUUUUUUUUUUUUUGGGG;(SEQ ID NO: 44) GGGUUUUUUUUUUUUUUUUUUGGG; (SEQ ID NO: 45)GUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUG; (SEQ ID NO: 46)GGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGG; (SEQ ID NO: 47)GGGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGGG; (SEQ ID NO: 48)GGGGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGGG; (SEQ ID NO: 49)GGGGGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGGGG; (SEQ ID NO: 50)GGGGGGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGGGGG; (SEQ ID NO: 51)GGGGGGGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGGGGGG; (SEQ ID NO: 52)GGGGGGGGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGGGGGGG; (SEQ ID NO: 53)GGGGGGGGGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGGGGGGGG; (SEQ ID NO: 54)GGUUUGG; (SEQ ID NO: 55) GGUUUUGG; (SEQ ID NO: 56) GGUUUUUGG;(SEQ ID NO: 57) GGUUUUUUGG; (SEQ ID NO: 58) GGUUUUUUUGG; (SEQ ID NO: 59)GGUUUUUUUUGG; (SEQ ID NO: 60) GGUUUUUUUUUGG; (SEQ ID NO: 61)GGUUUUUUUUUUGG; (SEQ ID NO: 62) GGUUUUUUUUUUUGG; (SEQ ID NO: 63)GGUUUUUUUUUUUUGG; (SEQ ID NO: 64) GGUUUUUUUUUUUUUGG; (SEQ ID NO: 65)GGUUUUUUUUUUUUUUGG; (SEQ ID NO: 66) GGUUUUUUUUUUUUUUUGG; (SEQ ID NO: 67)GGGUUUGGG; (SEQ ID NO: 68) GGGUUUUGGG; (SEQ ID NO: 69) GGGUUUUUGGG;(SEQ ID NO: 70) GGGUUUUUUGGG; (SEQ ID NO: 71) GGGUUUUUUUGGG;(SEQ ID NO: 72) GGGUUUUUUUUGGG; (SEQ ID NO: 73) GGGUUUUUUUUUGGG;(SEQ ID NO: 74) GGGUUUUUUUUUUGGG; (SEQ ID NO: 75) GGGUUUUUUUUUUUGGG;(SEQ ID NO: 76) GGGUUUUUUUUUUUUGGG; (SEQ ID NO: 77) GGGUUUUUUUUUUUUUGGG;(SEQ ID NO: 78) GGGUUUUUUUUUUUUUUUGGGUUUUUUUUUUUUUUUGGGUUUUUUUUUUUUUUUGGG; (SEQ ID NO: 79) GGGUUUUUUUUUUUUUUUGGGGGGUUUUUUUUUUUUUUUGGG;(SEQ ID NO: 80) GGGUUUGGGUUUGGGUUUGGGUUUGGGUUUGGGUUUGGGUUUGGGUUUG GG;

According to another particularly preferred embodiment, the problemunderlying the present invention may be solved by an alternative nucleicacid molecule according to formula (Ia)

(N_(u)C_(l)X_(m)C_(n)N_(v))_(a)

wherein:

-   C is cytidine (cytosine), uridine (uracil) or an analogue of    cytidine (cytosine) or uridine (uracil), preferably cytidine    (cytosine) or an analogue thereof-   X is guanosine (guanine), uridine (uracil), adenosine (adenine),    thymidine (thymine), cytidine (cytosine) or an analogue of the    above-mentioned nucleotides (nucleosides), preferably uridine    (uracil) or an analogue thereof-   N is each a nucleic acid sequence having independent from each other    a length of about 4 to 50, preferably of about 4 to 40, more    preferably of about 4 to 30 or 4 to 20 nucleic acids, each N    independently being selected from guanosine (guanine), uridine    (uracil), adenosine (adenine), thymidine (thymine), cytidine    (cytosine) or an analogue of these nucleotides (nucleosides);-   a is an integer from 1 to 20, preferably from 1 to 15, most    preferably from 1 to 10;-   l is an integer from 1 to 40,    -   wherein when l=1, C is cytidine (cytosine) or an analogue        thereof,        -   when l>1, at least 50% of these nucleotides (nucleosides)            are cytidine (cytosine) or an analogue        -   thereof;-   m is an integer and is at least 3;    -   wherein when m=3, X is uridine (uracil) or an analogue thereof,        when m>3, at least 3 successive uridines (uracils) or analogues        of uridine (uracil) occur;-   n is an integer from 1 to 40,    -   wherein when n=1, C is cytidine (cytosine) or an analogue        thereof,        -   when n>1, at least 50% of these nucleotides (nucleosides)            are cytidine (cytosine) or an analogue        -   thereof-   u, v may be independently from each other an integer from 0 to 50,    -   preferably wherein when u=0, v≧1, or        -   when v=0, u≧1;            wherein the nucleic acid molecule of formula (Ia) according            to the invention has a length of at least 50 nucleotides,            preferably of at least 100 nucleotides, more preferably of            at least 150 nucleotides, even more preferably of at least            200 nucleotides and most preferably of at least 250            nucleotides.

For formula (Ia), any of the definitions given above for elements N(i.e. N_(u) and N_(v)) and X (X_(m)), particularly the core structure asdefined above, as well as for integers a, l, m, n, u and v, similarlyapply to elements of formula (Ia) correspondingly, wherein in formula(Ia) the core structure is defined by C_(l)X_(m)C_(n). The definition ofbordering elements N_(u) and N_(v) is identical to the definitions givenabove for N_(u) and N_(v).

More particularly, C in the nucleic acid molecule of formula (Ia)according to the invention is a nucleotide or deoxynucleotide orcomprises a nucleoside, wherein the nucleotide (nucleoside) is typicallycytidine (cytosine) or uridine (uracil) or an analogue thereof. In thisconnection, cytidine (cytosine) or uridine (uracil) nucleotide analoguesare defined as non-natively occurring variants of naturally occurringcytidine (cytosine) or uridine (uracil) nucleotides. Accordingly,cytidine (cytosine) or uridine (uracil) analogues are chemicallyderivatized nucleotides (nucleosides) with non-natively occurringfunctional groups, which are preferably added to or deleted from thenaturally occurring cytidine (cytosine) or uridine (uracil) nucleotide(nucleoside) or which substitute the naturally occurring functionalgroups of a cytidine (cytosine) or uridine (uracil) nucleotide(nucleoside). Accordingly, each component of the naturally occurringcytidine (cytosine) or uridine (uracil) nucleotide may be modified,namely the base component, the sugar (ribose) component and/or thephosphate component forming the oligonucleotide's backbone. Thephosphate moieties may be substituted by e.g. phosphoramidates,phosphorothioates, peptide nucleotides, methylphosphonates etc., whereinthe naturally occurring phosphodiester backbone is still preferred.

Accordingly, analogues of cytidine (cytosine) or uridine (uracil)include, without implying any limitation, any naturally occurring ornon-naturally occurring cytidine (cytosine) or uridine (uracil) that hasbeen altered chemically, for example by acetylation, methylation,hydroxylation, etc., including, for example, 2-thio-cytidine (cytosine),3-methyl-cytidine (cytosine), 4-acetyl-cytidine (cytosine),dihydro-uridine (uracil), 4-thio-uridine (uracil),5-carboxymethylaminomethyl-2-thio-uridine (uracil),5-(carboxy-hydroxylmethyl)-uridine (uracil), 5-fluoro-uridine (uracil),5-bromo-uridine (uracil), 5-carboxymethylaminomethyl-uridine (uracil),5-methyl-2-thio-uridine (uracil), N-uridine (uracil)-5-oxyacetic acidmethyl ester, 5-methylaminomethyl-uridine (uracil),5-methoxyaminomethyl-2-thio-uridine (uracil),5′-methoxycarbonylmethyl-uridine (uracil), 5-methoxy-uridine (uracil),uridine (uracil)-5-oxyacetic acid methyl ester, uridine(uracil)-5-oxyacetic acid (v). The preparation of such analogues isknown to a person skilled in the art, for example from U.S. Pat. No.4,373,071, U.S. Pat. No. 4,401,796, U.S. Pat. No. 4,415,732, U.S. Pat.No. 4,458,066, U.S. Pat. No. 4,500,707, U.S. Pat. No. 4,668,777, U.S.Pat. No. 4,973,679, U.S. Pat. No. 5,047,524, U.S. Pat. No. 5,132,418,U.S. Pat. No. 5,153,319, U.S. Pat. No. 5,262,530 and U.S. Pat. No.5,700,642, the disclosures of which are incorporated by reference hereinin their entirety. In the case of an nucleotide (nucleoside) analogue asdescribed above, preference is given according to the inventionespecially to those analogues that increase the immunogenity of thenucleic acid molecule of formula (Ia) according to the invention and/ordo not interfere with a further modification that has been introduced.At least one cytidine (cytosine) or uridine (uracil) or an analoguethereof can occur in the core structure elements C_(l) and/or C_(n),optionally at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% 90% or even100% of the nucleotides (nucleosides) of the core structure elementsC_(l) and/or C_(n) are a naturally occurring cytidine (cytosine), anaturally occurring uridine (uracil), and/or an analogue thereof and/orexhibit properties of an analogue thereof as defined herein. Preferably,the core structure element C_(l) and/or C_(n) contains at least oneanalogue of a naturally occurring cytidine (cytosine) and/or a naturallyoccurring uridine (uracil) at all. Most preferably, all nucleotides(nucleosides) of these core structure elements C_(l) and/or C_(n) areanalogues, which may—most preferably—be identical analogues for the sametype of nucleotides (nucleosides) (e.g. all cytidine (cytosine)nucleotides are provided as 2-thio-cytidine (cytosine)) or they may bedistinct (e.g. at least two different cytidine (cytosine) analoguessubstitute the naturally occurring cytidine (cytosine) nucleotide).

The number of nucleotides (nucleosides) of core structure element C(C_(l) and/or C_(n)) in the nucleic acid molecule of formula (Ia)according to the invention is determined by l and n. l and n,independently of one another, are each an integer from 1 to 90, 1 to 80,1 to 70, 1 to 60, preferably 1 to 50, yet more preferably 1 to 40, andeven more preferably 1 to 30, wherein the lower limit of these rangesmay be 1, but alternatively also 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, oreven more. Preferably, for each integer, when l and/or n=1, C iscytidine (cytosine) or an analogue thereof, and when l or n>1, at least50%, more preferably at least 50%, 60%, 70%, 80%, 90% or even 100% ofthe nucleotides (nucleosides) of core structure element C (C_(l) and/orC_(n)) are cytidine (cytosine) or an analogue thereof. For example,without implying any limitation, when l or n=4, C_(l) and/or C_(n) canbe, for example, a CUCU, CCUU, UCUC, UUCC, CUUC, CCCU, CCUC, CUCC, UCCCor CCCC, etc.; when l or n=5, C_(l) and/or C_(n) can be, for example, aCCCUU, CCUCU, CUCCU, UCCCU, UCCUC, UCUCC, UUCCC, CUCUC, CCCCU, CCCUC,CCUCC, CUCCC, UCCCC, or CCCCC, etc.; etc. A nucleotide (nucleoside) ofcore structure elements C_(l) and/or C_(n) directly adjacent to X_(m) inthe nucleic acid molecule of formula (Ia) according to the invention ispreferably not an uridine (uracil) or an analogue thereof. Morepreferably nucleotides (nucleosides) of core structure elements C_(l)and/or C_(n) directly adjacent to X_(m) in the nucleic acid molecule offormula (Ia) according to the invention are at least one cytidine(cytosine) or an analogue thereof, more preferably a stretch of at least2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or even20 or more cytidines (cytosines) or an analogue thereof. Additionally, anucleotide (nucleoside) of core structure elements C_(l) and/or C_(n)directly adjacent to N, e.g. N_(u), and/or N_(v) (or N_(w1) or N_(w2) asdefined below) in the nucleic acid molecule of formula (Ia) according tothe invention is preferably not an uridine (uracil) or an analoguethereof. More preferably, nucleotides (nucleosides) of core structureelements C_(l) and/or C_(n) directly adjacent to N, e.g. N_(u), and/orN_(v) (or N_(w1) or N_(w2) as defined below) in the nucleic acidmolecule of formula (Ia) according to the invention are at least onecytidine (cytosine) or an analogue thereof, more preferably a stretch ofat least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19or even 20 or more cytidines (cytosines) or an analogue thereof.Likewise preferably, for formula (Ia), when l or n>1, at least 60%, 70%,80%, 90% or even 100% of the nucleotides of the core structure elementsC_(l) and/or C_(n) are cytidine (cytosine) or an analogue thereof, asdefined above. The remaining nucleotides (nucleosides) to 100% in thecore structure elements C_(l) and/or C_(n) (when cytidine (cytosine)constitutes less than 100% of these nucleotides (nucleosides)) may thenbe uridine (uracil) or an analogue thereof, as defined hereinbefore.

X, particularly X_(m), as a further core structure element in theinventive nucleic acid molecule according to formula (Ia), is preferablyas defined above for formula (I). The number of core structure element Xin the nucleic acid molecule of formula (Ia) according to the inventionis determined by m. m is an integer and is typically at least 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 20 to 30, 30 to40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to150, 150 to 200, or even more, wherein when m=3, X is uridine (uracil)or an analogue thereof, and when m>3, at least 3 or more directlysuccessive uridines (uracils) or an analogue thereof occur in theelement X of formula (Ia) above. Such a sequence of at least 3 or moredirectly successive uridines (uracils) is referred to in connection withthis application as a “monotonic uridine (uracil) sequence”. A monotonicuridine (uracil) sequence typically has a length of at least 3, 4, 5, 6,7, 8, 9 or 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 20 to 30, 30 to40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to150, 150 to 200 uridines (uracils) or optionally analogues of uridine(uracil) as defined above. Such a monotonic uridine (uracil) sequenceoccurs at least once in the core structure element X of the nucleic acidmolecule of formula (Ia) according to the invention. It is thereforepossible, for example, for 1, 2, 3, 4, 5 or more monotonic uridine(uracil) sequences having at least 3 or more uridines (uracils) oranalogues thereof to occur, which monotonic uridine (uracil) sequencescan be interrupted in the core structure element X by at least oneguanosine (guanine), adenosine (adenine), thymidine (thymine), cytidine(cytosine) or an analogue thereof, preferably 2, 3, 4, 5 or more. Forexample, when m=3, X_(m) is a UUU. When m=4, X_(m) can be, for example,without implying any limitation, a UUUA, UUUG, UUUC, UUUU, AUUU, GUUU orCUUU, etc. When n=10, X_(m) can be, for example, without implying anylimitation, a UUUAAUUUUC (SEQ ID NO: 120), UUUUGUUUUA (SEQ ID NO: 121),UUUGUUUGUU (SEQ ID NO: 122), UUGUUUUGUU (SEQ ID NO: 123), UUUUUUUUUU(SEQ ID NO: 124), etc. The nucleotides (nucleosides) of X_(m) adjacentto C_(l) or C_(n) of the nucleic acid molecule of formula (Ia) accordingto the invention preferably comprise uridine (uracil) or analoguesthereof. When m>3, typically at least 50%, preferably at least 60%, 70%,80%, 90% or even 100%, of the nucleotides of X_(m) are uridine (uracil)or an analogue thereof, as defined above. The remaining nucleotides(nucleosides) of X_(m) to 100% (where there is less than 100% uridine(uracil) in the sequence X_(m)) may then be guanosine (guanine), uridine(uracil), adenosine (adenine), thymidine (thymine), cytidine (cytosine)or an analogue thereof, as defined above.

Likewise, the inventive nucleic acid according formula (Ia) abovecontains a bordering element N, particularly N_(u) and/or N_(v), whereinthe bordering element N, particularly N_(u) and/or N_(v), as well asintegers x and y are as defined above.

The element N_(u)C_(l)X_(m)C_(n)N_(v) may occur as a repetitive elementaccording to the inventive nucleic acid molecule of formula (Ia)(N_(u)C_(l)X_(m)C_(n)N_(v))_(a), as defined above, wherein the number ofrepetitions of this element according to formula (Ia)(N_(u)C_(l)X_(m)C_(n)N_(v))_(a) is determined by the integer a.Preferably, a is an integer from about 1 to 100, 1 to 50, 1 to 20, morepreferably an integer from about 1 to 15, most preferably an integerfrom about 1 to 10. In this context, the repetitive elementsN_(u)C_(l)X_(m)C_(n)N_(v) may be equal or different from each other.

According to a particularly preferred embodiment, the inventive moleculeof formula (Ia) (N_(u)C_(l)X_(m)C_(n)N_(v))_(a), as defined above,comprises a core structure C_(l)X_(m)C_(n), preferably selected from atleast one of the following sequences of SEQ ID NOs: 81-83:

(SEQ ID NO: 81) CCCUUUUUUUUUUUUUUUCCCUUUUUUUUUUUUUUUCCCUUUUUUUUUUUUUUUCCC (SEQ ID NO: 82)CCCUUUCCCUUUCCCUUUCCCUUUCCCUUUCCCUUUCCCUUUCCCUUUCC  C (SEQ ID NO: 83)CCCUUUUUUUUUUUUUUUCCCCCCUUUUUUUUUUUUUUUCCC 

The inventive nucleic acid molecule according to either formula (I) (or(Ia)), particularly each single repetitive elementN_(u)G_(l)X_(m)G_(n)N_(v) (or N_(u)C_(l)X_(m)C_(n)N_(v)) thereof, may besingle-stranded, double-stranded or partially double-stranded, etc. asdefined for formula (I) in general.

If the inventive nucleic acid molecule according to either formula (I)(or (Ia)) is a single-stranded nucleic acid molecule, the sequence istypically single-stranded over its entire length.

Likewise, if the inventive nucleic acid molecule according to eitherformula (I) (or (Ia)) is a double-stranded nucleic acid molecule, thesequence is typically double-stranded over its entire length.

If the inventive nucleic acid molecule according to either formula (I)(or (Ia)) is a partially double-stranded nucleic acid molecule, thenucleic acid sequence of a nucleic acid molecule of either formula (I)(or (Ia)) may be single-stranded in the region outside the corestructure G_(l)X_(m)G_(n) (or C_(l)X_(m)C_(n)), and double-stranded inthe region of said core structure, the core structure G_(l)X_(m)G_(n)(or C_(l)X_(m)C_(n)), preferably being selected from at least one of theabove defined sequences of SEQ ID NOs: 1-83. Even more preferably, thecore structure G_(l)X_(m)G_(n) (or C_(l)X_(m)C_(n)) of (either) formula(I) (or (Ia)) may be double-stranded in such a region of the corestructure, wherein a stretch of uridines (uracils) occurs, mostpreferably over the entire uridine (uracil) stretch or at least 60%,70%, 80%, 90%, 95%, 98% or 99% thereof.

Alternatively or additionally, if the inventive nucleic acid moleculeaccording to either formula (I) or (Ia) is a partially double-strandednucleic acid molecule, other parts (than the core structureG_(l)X_(m)G_(n)) of the inventive nucleic acid molecule according toformula (I) or (Ia) as defined above may be double-stranded. E.g., thenucleic acid sequence of a nucleic acid molecule of either formula (I)or (Ia) may be double-stranded in the region outside the core structureG_(l)X_(m)G_(n) (or C_(l)X_(m)C_(n)), e.g. in the bordering elementsN_(u) and/or N_(v), and single-stranded in the region of said corestructure, the core structure G_(l)X_(m)G_(n) (or C_(l)X_(m)C_(n)),preferably selected from at least one of the above defined sequences ofSEQ ID NOs: 1-83. E.g. at least one of the bordering elements N_(v)and/or N_(v) may be double-stranded, whereas the remaining elements ofeither formula (I) or (Ia), e.g. the core structure G_(l)X_(m)G_(n)and/or other elements, may remain single-stranded.

Alternatively or additionally, the inventive nucleic acid moleculeaccording to formula (I) may be selected from a mixture of asingle-stranded nucleic acid molecule according to either formula (I) or(Ia) and a (partially) double-stranded nucleic acid molecule accordingto either formula (I) (or (Ia)), preferably in a ratio of about 1:10 to10:1, more preferably in a ratio of 1:3 to 3:1.

According to a very particularly preferred embodiment, the inventivenucleic acid molecule according to formula (I) may be selected from e.g.any of the following sequences:

from SEQ ID NO: 84: UAGCGAAGCU CUUGGACCUA GG UUUUU UUUUU UUUUU GGG UGCGUUCCUA GAAGUACACG or from SEQ ID NO: 85:UAGCGAAGCU CUUGGACCUA GG UUUUU UUUUU UUUUU GGG UGCGUUCCUA GAAGUACACG AUCGCUUCGA GAACCUGGAU CCAAAAA AAAAA AAAAA CCC ACGCAAGGAU CUUCAUGUGCor from SEQ ID NO: 114 (R820: (N₁₀₀)₂)GGGAGAAAGCUCAAGCUUGGAGCAAUGCCCGCACAUUGAGGAAACCGAGUUGCAUAUCUCAGAGUAUUGGCCCCCGUGUAGGUUAUUCUUGACAGACAGUGGAGCUUAUUCACUCCCAGGAUCCGAGUCGCAUACUACGGUACUGGUGACAGACCUAGGUCGUCAGUUGACCAGUCCGCCACUAGACGUGAGUCCGUCAAAGCAGUUAGAUGUUACACUCUAUUAGAUC or from SEQ ID NO: 115 (R719: (N₁₀₀)₅)GGGAGAAAGCUCAAGCUUGGAGCAAUGCCCGCACAUUGAGGAAACCGAGUUGCAUAUCUCAGAGUAUUGGCCCCCGUGUAGGUUAUUCUUGACAGACAGUGGAGCUUAUUCACUCCCAGGAUCCGAGUCGCAUACUACGGUACUGGUGACAGACCUAGGUCGUCAGUUGACCAGUCCGCCACUAGACGUGAGUCCGUCAAAGCAGUUAGAUGUUACACUCUAUUAGAUCUCGGAUUACAGCUGGAAGGAGCAGGAGUAGUGUUCUUGCUCUAAGUACCGAGUGUGCCCAAUACCCGAUCAGCUUAUUAACGAACGGCUCCUCCUCUUAGACUGCAGCGUAAGUGCGGAAUCUGGGGAUCAAAUUACUGACUGCCUGGAUUACCCUCGGACAUAUAACCUUGUAGCACGCUGUUGCUGUAUAGGUGACCAACGCCCACUCGAGUAGACCAGCUCUCUUAGUCCGGACAAUGAUAGGAGGCGCGGUCAAUCUACUUCUGGCUAGUUAAGAAUAGGCUGCACCGACCUCUAUAAGUAGCGUGUCCUCUAGor from SEQ ID NO: 116 (R720: (N₁₀₀)₁₀)GGGAGAAAGCUCAAGCUUGGAGCAAUGCCCGCACAUUGAGGAAACCGAGUUGCAUAUCUCAGAGUAUUGGCCCCCGUGUAGGUUAUUCUUGACAGACAGUGGAGCUUAUUCACUCCCAGGAUCCGAGUCGCAUACUACGGUACUGGUGACAGACCUAGGUCGUCAGUUGACCAGUCCGCCACUAGACGUGAGUCCGUCAAAGCAGUUAGAUGUUACACUCUAUUAGAUCUCGGAUUACAGCUGGAAGGAGCAGGAGUAGUGUUCUUGCUCUAAGUACCGAGUGUGCCCAAUACCCGAUCAGCUUAUUAACGAACGGCUCCUCCUCUUAGACUGCAGCGUAAGUGCGGAAUCUGGGGAUCAAAUUACUGACUGCCUGGAUUACCCUCGGACAUAUAACCUUGUAGCACGCUGUUGCUGUAUAGGUGACCAACGCCCACUCGAGUAGACCAGCUCUCUUAGUCCGGACAAUGAUAGGAGGCGCGGUCAAUCUACUUCUGGCUAGUUAAGAAUAGGCUGCACCGACCUCUAUAAGUAGCGUGUCCUCUAGAGCUACGCAGGUUCGCAAUAAAAGCGUUGAUUAGUGUGCAUAGAACAGACCUCUUAUUCGGUGAAACGCCAGAAUGCUAAAUUCCAAUAACUCUUCCCAAAACGCGUACGGCCGAAGACGCGCGCUUAUCUUGUGUACGUUCUCGCACAUGGAAGAAUCAGCGGGCAUGGUGGUAGGGCAAUAGGGGAGCUGGGUAGCAGCGAAAAAGGGCCCCUGCGCACGUAGCUUCGCUGUUCGUCUGAAACAACCCGGCAUCCGUUGUAGCGAUCCCGUUAUCAGUGUUAUUCUUGUGCGCACUAAGAUUCAUGGUGUAGUCGACAAUAACAGCGUCUUGGCAGAUUCUGGUCACGUGCCCUAUGCCCGGGCUUGUGCCUCUCAGGUGCACAGCGAUACUUAAAGCCUUCAAGGUACUCGACGUGGGUACCGAUUCGUGACACUUCCUAAGAUUAUUCCACUGUGUUAGCCCCGCACCGCCGACCUAAACUGGUCCAAUGUAUACGCAUUCGCUGAGCGGAUCGAUAAUAAAAGCUUGAAUUor from SEQ ID NO: 117 (R821: (N₄₀U₂₀N₄₀)₂)GGGAGAAAGCUCAAGCUUAUCCAAGUAGGCUGGUCACCUGUACAACGUAGCCGGUAUUUUUUUUUUUUUUUUUUUUUUGACCGUCUCAAGGUCCAAGUUAGUCUGCCUAUAAAGGUGCGGAUCCACAGCUGAUGAAAGACUUGUGCGGUACGGUUAAUCUCCCCUUUUUUUUUUUUUUUUUUUUUAGUAAAUGCGUCUACUGAAUCCAGCGAUGAUGCUGGCCCAGAUCor from SEQ ID NO: 118 (Seq. R722: (N₄₀U₂₀N₄₀)₅)GGGAGAAAGCUCAAGCUUAUCCAAGUAGGCUGGUCACCUGUACAACGUAGCCGGUAUUUUUUUUUUUUUUUUUUUUUUGACCGUCUCAAGGUCCAAGUUAGUCUGCCUAUAAAGGUGCGGAUCCACAGCUGAUGAAAGACUUGUGCGGUACGGUUAAUCUCCCCUUUUUUUUUUUUUUUUUUUUUAGUAAAUGCGUCUACUGAAUCCAGCGAUGAUGCUGGCCCAGAUCUUCGACCACAAGUGCAUAUAGUAGUCAUCGAGGGUCGCCUUUUUUUUUUUUUUUUUUUUUUUGGCCCAGUUCUGAGACUUCGCUAGAGACUACAGUUACAGCUGCAGUAGUAACCACUGCGGCUAUUGCAGGAAAUCCCGUUCAGGUUUUUUUUUUUUUUUUUUUUUCCGCUCACUAUGAUUAAGAACCAGGUGGAGUGUCACUGCUCUCGAGGUCUCACGAGAGCGCUCGAUACAGUCCUUGGAAGAAUCUUUUUUUUUUUUUUUUUUUUUUGUGCGACGAUCACAGAGAACUUCUAUUCAUGCAGGUCUGCUCUAor from SEQ ID NO: 119 (R723: (N₄₀U₂₀N₄₀)₁₀):GGGAGAAAGCUCAAGCUUAUCCAAGUAGGCUGGUCACCUGUACAACGUAGCCGGUAUUUUUUUUUUUUUUUUUUUUUUGACCGUCUCAAGGUCCAAGUUAGUCUGCCUAUAAAGGUGCGGAUCCACAGCUGAUGAAAGACUUGUGCGGUACGGUUAAUCUCCCCUUUUUUUUUUUUUUUUUUUUUAGUAAAUGCGUCUACUGAAUCCAGCGAUGAUGCUGGCCCAGAUCUUCGACCACAAGUGCAUAUAGUAGUCAUCGAGGGUCGCCUUUUUUUUUUUUUUUUUUUUUUUGGCCCAGUUCUGAGACUUCGCUAGAGACUACAGUUACAGCUGCAGUAGUAACCACUGCGGCUAUUGCAGGAAAUCCCGUUCAGGUUUUUUUUUUUUUUUUUUUUUCCGCUCACUAUGAUUAAGAACCAGGUGGAGUGUCACUGCUCUCGAGGUCUCACGAGAGCGCUCGAUACAGUCCUUGGAAGAAUCUUUUUUUUUUUUUUUUUUUUUUGUGCGACGAUCACAGAGAACUUCUAUUCAUGCAGGUCUGCUCUAGAACGAACUGACCUGACGCCUGAACUUAUGAGCGUGCGUAUUUUUUUUUUUUUUUUUUUUUUUCCUCCCAACAAAUGUCGAUCAAUAGCUGGGCUGUUGGAGACGCGUCAGCAAAUGCCGUGGCUCCAUAGGACGUGUAGACUUCUAUUUUUUUUUUUUUUUUUUUUUCCCGGGACCACAAAUAAUAUUCUUGCUUGGUUGGGCGCAAGGGCCCCGUAUCAGGUCAUAAACGGGUACAUGUUGCACAGGCUCCUUUUUUUUUUUUUUUUUUUUUUUCGCUGAGUUAUUCCGGUCUCAAAAGACGGCAGACGUCAGUCGACAACACGGUCUAAAGCAGUGCUACAAUCUGCCGUGUUCGUGUUUUUUUUUUUUUUUUUUUUGUGAACCUACACGGCGUGCACUGUAGUUCGCAAUUCAUAGGGUACCGGCUCAGAGUUAUGCCUUGGUUGAAAACUGCCCAGCAUACUUUUUUUUUUUUUUUUUUUUCAUAUUCCCAUGCUAAGCAAGGGAUGCCGCGAGUCAUGUUAAGCUUGAAUU

According to another very particularly preferred embodiment, theinventive nucleic acid molecule according to formula (Ia) may beselected from e.g. any of the following sequences:

(SEQ ID NO: 86) UAGCGAAGCU CUUGGACCUA CC UUUUU UUUUU UUUUU CCC UGCGUUCCUA GAAGUACACG or (SEQ ID NO: 87)UAGCGAAGCU CUUGGACCUA CC UUUUU UUUUU UUUUU CCC UGCGUUCCUA GAAGUACACG AUCGCUUCGA GAACCUGGAU GG AAAAA AAAAA AAAAA GGG ACGCAAGGAU CUUCAUGUGC

According to one preferred embodiment, the inventive nucleic acidmolecule according to formula (I) (or (Ia)) as defined above may bemodified with a poly(X) sequence (modifying element). Such inventivenucleic acid molecules may comprise e.g. a nucleic acid moleculeaccording to formula (II):

poly(X)_(s)(N_(u)G_(l)X_(m)G_(n)N_(v))_(a)poly(X)_(t),

wherein the nucleic acid molecule of formula (II) according to theinvention likewise has a length of at least 50 nucleotides, preferablyof at least 100 nucleotides, more preferably of at least 150nucleotides, even more preferably of at least 200 nucleotides and mostpreferably of at least 250 nucleotides.

In a nucleic acid molecule according to inventive formula (II), theelements G, X and N, particularly, the core structure G_(l)X_(m)G_(n),and the elements N_(u) and N_(v), as well as the integers a, l, m, n, uand v are as defined above for formula (I). In the context of thepresent invention, a modifying element poly(X), particularly poly(X)_(s)and/or poly(X)_(t), of an inventive nucleic acid molecule according toformula (II), is typically a single-stranded, a double-stranded or apartially double-stranded nucleic acid sequence, e.g a DNA or RNAsequence as defined above in general. Preferably, the modifying elementpoly(X), particularly poly(X)_(s) and/or poly(X)_(t), is a homopolymericstretch of nucleic acids, wherein X may be any nucleotide ordeoxynucleotide or comprises a nucleoside as defined above for X of aninventive nucleic acid molecule according to formula (I) or (Ia).Preferably, X may selected independently for each poly(X), particularlypoly(X)_(s) and/or poly(X)_(t), from a nucleotide or deoxynucleotide orcomprises a nucleoside, wherein the nucleotide (nucleoside) is selectedfrom guanosine (guanine), uridine (uracil), adenosine (adenine),thymidine (thymine), cytidine (cytosine), inosine or an analogue ofthese nucleotides, e.g. from a single-stranded stretch of cytidines(cytosines) (poly(C)), of guanosine (guanine)s (poly(G)), of adenosine(adenine)s (poly(A)), of uridines (uracils) (poly(U)), of inosines(poly(I)), etc. or from a homopolymeric double-stranded stretch ofinosines and cytidines (cytoines) (poly(I:C)), of adenosine (adenine)and uridines (uracils) (poly(A:U)), etc., wherein the homoplymericsequence, particularly poly(I:C) and/or poly(A:U), may be coupled to thesequence (N_(u)G_(l)X_(m)G_(n)N_(v))_(a) of the nucleic acid moleculeaccording to formula (II) via any of its strands, e.g. either using thepoly-C, the poly-I, the poly-A or the poly-U sequence. The length ofmodifying element poly(X), particularly poly(X)_(s) and/or poly(X)_(t),of the nucleic acid molecule of inventive formula (II) is determined byintegers s and/or t, wherein s and/or t, independent from each other,may be an integer from about 5 to 100, preferably about 5 to 70, morepreferably about 5 to 50, even more preferably about 5 to 30 and mostpreferably about 5 to 20.

According to a particularly preferred embodiment, a nucleic acidmolecule according to formula (II) as defined above, may specificallycomprise e.g. a nucleic acid molecule according to formula (IIa),

poly(X)(N_(u)G_(l)X_(m)G_(n)N_(v))_(n),

or a nucleic acid molecule according to formula (IIb),

poly(X)(N_(u)G_(l)X_(m)G_(n)N_(v))_(a)poly(X),

wherein any of these nucleic acid molecules of formulas (IIa) or (IIb)according to the invention likewise has a length of at least 50nucleotides, preferably of at least 100 nucleotides, more preferably ofat least 150 nucleotides, even more preferably of at least 200nucleotides and most preferably of at least 250 nucleotides. Similarly,all other definitions apply as set forth for formula (II) or (I) above.Likewise, said formulas (II), (IIa) and (IIb) may be defined on basis ofa formula according to formula (Ib), i.e. introducing the core structureC_(l)X_(m)C_(n).

More preferably, poly(X) in an inventive nucleic acid molecule accordingto either formula (II), (IIa) and/or (IIb) may be selected from apoly(X) as defined above, more preferably from poly(I:C) and/or frompoly(A:U). These modifying elements poly(X), particularly poly(I:C)and/or poly(A:U), may be coupled to the sequence according to formula(II), (IIa) and/or (IIb) via any of its strands, e.g. either using thepoly-C, the poly-G, the poly-I, the poly-A or the poly-U sequence.

Similarly as defined above for formula (I) or (Ia), the inventivenucleic acid molecule according to either formula (II), (IIa) and/or(IIb) may be a single-stranded, a double-stranded or a partiallydouble-stranded nucleic acid molecule, as defined above.

If the inventive nucleic acid molecule according to either formula (II),(IIa) and/or (IIb) is a single-stranded nucleic acid molecule, thesequence is typically single-stranded over its entire length.

Likewise, if the inventive nucleic acid molecule according to eitherformula (II), (IIa) and/or (IIb) is a double-stranded nucleic acidmolecule, the sequence is typically double-stranded over its entirelength.

If the inventive nucleic acid molecule according to either formula (II),(IIa) and/or (IIb) is a partially double-stranded nucleic acid molecule,the nucleic acid sequence of a nucleic acid molecule of either formula(II), (IIa) and/or (IIb) may be single-stranded in the region outsidethe core structure G_(l)X_(m)G_(n), and double-stranded in the region ofsaid core structure, the core structure G_(l)X_(m)G_(n), preferablybeing selected from at least one of the above defined sequences of SEQID NOs: 1-80 or SEQ ID NOs: 81 to 83. Even more preferably, the corestructure G_(l)X_(m)G_(n) (or C_(l)X_(m)C_(n)) of either formula (I) (or(Ia)) may be double-stranded in such a region of the core structure,wherein a stretch of uridines (uracils) occurs, most preferably over theentire uridine (uracil) stretch or at least 60%, 70%, 80%, 90%, 95%, 98or 99% thereof.

Alternatively or additionally, if the inventive nucleic acid moleculeaccording to either formula (II), (IIa) and/or (IIb) is a partiallydouble-stranded nucleic acid molecule, other parts (than the corestructure G_(l)X_(m)G_(n)) of the inventive nucleic acid moleculeaccording to either formula (II), (IIa) and/or (IIb) as defined abovemay be double-stranded. E.g., the nucleic acid sequence of a nucleicacid molecule of either formula (II), (IIa) and/or (IIb) may bedouble-stranded in the region outside the core structureG_(l)X_(m)G_(n), e.g. in the bordering elements N_(u) and/or N_(v),and/or in the modifying element poly(X), e.g. poly(X)_(s) and orpoly(X)_(t) (such as e.g. a poly(I:C) or poly(A:U) sequence), and e.g.single-stranded in the region of said core structure, the core structureG_(l)X_(m)G_(n), preferably being selected from at least one of theabove defined sequences of SEQ ID NOs: 1-83. E.g. at least one of thebordering elements N_(u) and/or N_(v), and/or at least one of themodifying elements poly(X), e.g. poly(X)_(s) and or poly(X)_(t), may bedouble-stranded, whereas the remaining elements of either formula (II),(IIa) and/or (IIb), e.g. the core structure G_(l)X_(m)G_(n) and/or otherelements, may remain single-stranded.

Alternatively or additionally a mixture of a single-stranded nucleicacid molecule according to either formula (II), (IIa)) and/(IIb) and a(partially) double-stranded nucleic acid molecule according to eitherformula (II), (IIa)) and/(IIb), preferably in a ratio of about 1:10 to10:1, more preferably in a ratio of 1:3 to 3:1.

According to a particularly preferred embodiment, the inventive nucleicacid molecule according to either formula (II), (IIa) and/or (IIb) maybe selected from e.g. any of the following sequences:

(SEQ ID NO: 88) CCCCCCCCCC CCCCCCCCCCGG UUUUU UUUUU UUUUU GGG(SEQ ID NO: 89) CCCCCCCCCC CCCCCCCCCCGG UUUUU UUUUU UUUUU GGGIIIIIIIIII IIIIIIIIIIII (SEQ ID NO: 90)CCCCCCCCCC CCCCCCCCCCGG UUUUU UUUUU UUUUU GGG                        AAAAA AAAAA AAAAA (SEQ ID NO: 91)CCCCCCCCCC CCCCCCCCCC GG UUUUU UUUUU UUUUU GGGGGGGGGGGGG GGGGGGGGGG CC AAAAA AAAAA AAAAA CCC (SEQ ID NO: 92)CCCCCCCCCC CCCCCCCCCC UAGCGAAGCU CUUGGACCUA GGUUUUU UUUUU UUUUU GGG UGCGUUCCUA GAAGUACACG (SEQ ID NO: 93)CCCCCCCCCC CCCCCCCCCC GG UUUUU UUUUU UUUUU GGGUGCGUUCCUA GAAGUACACG GGGGGGGGGG GGGGGGGGGG CCAAAAA AAAAA AAAAA CCC ACGCAAGGAU CUUCAUGUGC UAGCGAAGCU CUUGGACCUA AUCGCUUCGA GAACCUGGAU (SEQ ID NO: 94)CCCCCCCCCC CCCCCCCCCC GG UUUUU UUUUU UUUUU GGGUGCGUUCCUA GAAGUACACG CC AAAAA AAAAA AAAAA CCCACGCAAGGAU CUUCAUGUGC UAGCGAAGCU CUUGGACCUA  AUCGCUUCGA GAACCUGGAU

According to a further preferred embodiment, an inventive nucleic acidmolecule according to formula (I) (or (Ia)) as defined above may bemodified by inserting a stem or a stem loop, e.g. leading to a nucleicacid molecule according to formula (IIIc),

(N_(u)stem1G_(l)X_(m)G_(n)stem2N_(v))_(a),

or to a nucleic acid molecule according to formula (IIIb),

(N_(u)G_(l)X_(m)G_(n)N_(v))_(a)stem1N_(w1)stem2N_(w3),

wherein the nucleic acid molecule of either formula (IIIc) and/or (IIIb)according to the invention has a length of at least 100 nucleotides,more preferably of at least 150 nucleotides, even more preferably of atleast 200 nucleotides and most preferably of at least 250 nucleotides.Likewise, said formulas (IIIc) and (IIIb) may be defined on basis of aformula according to formula (Ib), i.e. introducing the core structureC_(l)X_(m)C_(n).

Particularly, the inventive nucleic acids of either formula (IIIc)and/or (IIIb) represent variants of formula (I) as defined above. In anucleic acid according to any of formulas (IIIc) and/or (IIIb), thebordering elements N, i.e. N_(u) and/or N_(v), bordering the corestructure G_(l)X_(m)G_(n), are further augmented by at least one stem orstem loop structure, preferably consisting of single stem loop elementsstem1 and stem2. In the inventive nucleic acids according to any offormulas (IIIc) and/or (IIIb) as defined above, the elements G, X and N,particularly, the core structure G_(l)X_(m)G_(n), and the integers a, l,m, n, u and v are as defined above. More preferably integer a=1.Optionally u and/or v may be 0. Additionally, elements N_(w1) andN_(w2), adjacent to stem loop elements stem1 and stem2, representfurther bordering elements, which are defined as described above forbordering elements N_(u) and/or N_(v). Particularly, bordering element Nin general is as described above for N in formula (I) above, andintegers w1 and w2 are independently selected from each other and aredefined as above in formula (I) for integers u and/or v.

In this context, a stem or stem loop structure is an intramolecular basepairing that can occur in single-stranded DNA or, more commonly, in RNA.The structure is also known as a hairpin or hairpin loop. It occurs whentwo regions of the same molecule, e.g. stem loop elements stem1 andstem2, usually palindromic sequence elements in nucleic acid sequences,form base-pairs with each other, leading to (a double helix that endsin) an unpaired loop. The unpaired loop thereby typically represents aregion of the nucleic acid, which shows no or nearly no homology withthe sequence of either stem1 or stem2 and is thus not capable of basepairing with any of these stem loop elements. The resultinglollipop-shaped structure is a key building block of many RNA secondarystructures. The formation of a stem-loop structure is thus dependent onthe stability of the resulting helix and loop regions, wherein the firstprerequisite is typically the presence of a sequence that can fold backon itself to form a paired double helix. The stability of paired stemloop elements is determined by the length, the number of mismatches orbulges it contains (a small number of mismatches is typically tolerable,especially in a long helix), and the base composition of the pairedregion. E.g., pairings between guanosine (guanine) and cytidine(cytosine) may be more preferred in such sequences, since they havethree hydrogen bonds and are more stable compared to adenosine(adenine)-uridine (uracil) pairings, which have only two. In RNA,guanosine (guanine)-uridine (uracil) pairings featuring two hydrogenbonds may thus be favorable. The stability of the loop also influencesthe formation of the stem-loop structure. “Loops” (i.e. only the loopnot containing stem loop elements stem1 and stem2) that are less thanthree bases long are sterically less preferable. However, stems, i.e.formations which show no (defined) loop but just an unpaired regionbetween stem1 and stem2 may also be included. In the context of thepresent invention, optimal loop length tends to be about 4-100 baseslong, more preferably 4 to 50 or even 4 to 30 or even 4 to 20 bases.

Hence, in the context of a nucleic acid molecule according to any offormulas (IIIc) and/or (IIIb), stem loop elements stem1 and stem2typically represent parts of one stem or stem loop structure, whereinthe stem or stem loop structure may be formed by stem loop elementsstem1 and stem2, and a loop may be formed by a sequence, which islocated between these stem loop elements. The stem or stem loop may havethe form of a helix in the base-paired region. Each stem loop elementstem1 and stem2, is preferably a nucleic acid as defined above, morepreferably an RNA, and most preferably a single-stranded RNA, whereinany of nucleotides (nucleosides) or analogs as defined above for corestructure element X may be used as a nucleotides (nucleosides) foreither stem1 and/or stem2. Additionally, stem loop element stem1represents a palindromic sequence of stem loop element stem2. Bothsequences are therefore preferably capable of base pairing with eachother and thus together form basis for a stem or stem loop.

Therefore, stem loop elements stems or stem2 may be selected pairwisefrom any nucleic acid sequence, provided that stem loop elements stem1or stem2 are palindromic to each other, i.e. that one sequence is equalto the other (complementary) sequence read backwards or shows a homologyto this sequence of at least 90%, more preferably of at least 95%, andmost preferably of at least 99% to the other sequence, when readbackwards. Such palindromic sequences stem1 and stem2 may be formed eachby a nucleic acid sequence having a length of about 5 to 50, morepreferably about 5 to 40 and most preferably about 5 to 30 nucleicacids, selected from adenosine (adenine), guanosine (guanine), cytidine(cytosine), uridine (uracil), thymidine (thymine), or an analoguethereof as defined herein.

Exemplary sequences for stem loop elements stem1 and stem2 may includee.g.:

a) for stem1: (SEQ ID NO: 95) UAGCGAAGCUCUUGGACCUA  for stem2:(SEQ ID NO: 96) UAGGUCCAAGAGCUUCGCUA  b) for stem1: (SEQ ID NO: 96)UAGGUCCAAGAGCUUCGCUA  for stem2: (SEQ ID NO: 95) UAGCGAAGCUCUUGGACCUA c) for stems: (SEQ ID NO: 97) GCCGCGGGCCG  for stem2: (SEQ ID NO: 98)CGGCCCGCGGC  d) for stem1: (SEQ ID NO: 98) CGGCCCGCGGC  for stem2:(SEQ ID NO: 97) GCCGCGGGCCG  e) for stem1: (SEQ ID NO: 99) GACACGGUGC for stem2: (SEQ ID NO: 100) GCACCGUGCA  f) for stem1: (SEQ ID NO: 100)GCACCGUGCA  for stem2: (SEQ ID NO: 99) GACACGGUGC  g) for stem1:(SEQ ID NO: 101) ACCUAGGU  for stem2: (SEQ ID NO: 101) ACCUAGGU h) for stem1: (SEQ ID NO: 102) UGGAUCCA  for stem2: (SEQ ID NO: 102)UGGAUCCA  i) for stem1: (SEQ ID NO: 103) CCUGC  for stem2:(SEQ ID NO: 104) GCAGG  j) for stem1: (SEQ ID NO: 105) GCAGG  for stem2:(SEQ ID NO: 106) CCUGC  etc.

According to one first alternative, the core structure G_(l)X_(m)G_(n)may be located within the stem loop structure, i.e. the core structureG_(l)X_(m)G_(n) may be located between stem loop elements stem1 andstem2, thereby preferably forming a loop. Such a nucleic acid moleculeis resembled by formula (IIIc), having the composition (N_(u) stem1G_(l)X_(m)G_(n) stem2 N_(v))_(a), as defined above. When u and/or v=0,and a=1 formula (IIIc) may lead to a specific nucleic acid molecule“stem1 G_(l)X_(m)G_(n) stem2”, which is also incorporated by the presentinvention.

According to another alternative, the core structure G_(l)X_(m)G_(n) maybe located outside the stem loop structure, wherein likewise stem loopelements stem1 and stem2 may be separated from each other by a sequence,preferably a bordering element N, e.g. N_(w1) or N_(w2), which then mayform a loop structure upon base pairing of stem loop elements stem1 andstem2. Additionally, stem loop elements 1 and/or 1, adjacent to the corestructure G_(l)X_(m)G_(n) may be separated from the core structureG_(l)X_(m)G_(n) by a further bordering element, e.g. N_(w1) or N_(w2).According to the present invention, such a nucleic acid is resembled byformula (Mb), having the composition (N_(u)G_(l)X_(m)G_(n)N_(v))_(a)stem1 N_(w1) stem2 N_(w2), as defined above.

The inventive nucleic acid molecule according to either formula (IIIc)and/or (IIIb) may be single-stranded, or partially double-stranded.

If the inventive nucleic acid molecule according to either formula(IIIc) and/or (IIIb) is a single-stranded nucleic acid molecule, thesequence is typically single-stranded over its entire length.

If the inventive nucleic acid molecule according to either formula(IIIc) and/or (IIIb) is a partially double-stranded nucleic acidmolecule, the nucleic acid molecule of either formula (IIIc) and/or(IIIb) preferably may be single-stranded in the region of the stem loopelements stem1 and stem2 and in the regions of the loop formed by eitherthe core structure G_(l)X_(m)G_(n) or by any other element, e.g. N_(w1)or N_(w2). Elements positioned outside the stem loop elements stem1 andstem2 and in the regions of the loop formed by either the core structureG_(l)X_(m)G_(n) or by any other element, e.g. N_(w1) or N_(w2), may thenbe, independent from each other, single or double-stranded.

Alternatively or additionally a mixture of a single-stranded orpartially double-stranded nucleic acid molecule according to eitherformula (IIIc) or (IIIb) and a (partially) double-stranded nucleic acidmolecule according to either formula (IIIc) or (IIIb), preferably in aratio of about 1:10 to 10:1, more preferably in a ratio of 1:3 to 3:1.

According to a very particularly preferred embodiment, the inventivenucleic acid molecule according to either formula (IIIc) and/or (IIIb),respectively, may be selected from e.g. any of the following sequences:

(SEQ ID NO: 107) UAGCGAAGCU CUUGGACCUA GG UUUUU UUUUU UUUUU GGG UAGGUCCAAG AGCUUCGCUA (SEQ ID NO: 108)UAGCGAAGCU CUUGGACCUA GG UUUUU UUUUU UUUUU GGG UGCGUUCCUA GAAGUACACG GCCGCGGGCCG UGCGUUCCUA GAAGUACACG CGGCCCGCGGC UGCGUUCCUA GAAGUACACG(stem1 and stem2 are underlined, the core structure G_(l)X_(m)G_(n) iswritten in bold)

Nucleic acid molecules of either formula (I), (Ia), (II), (IIa), (IIb),(IIIc) and/or (IIIb) according to the invention as defined above, may beprepared using any method known in the art, including synthetic methodssuch as e.g. solid phase synthesis, as well as in vitro methods such asin vitro transcription reactions. Preferably, an in vitro transcriptionis used for preparation of the inventive nucleic acid molecules. Assurprisingly found by the inventors of the present invention, nucleicacid molecules of either formula (I), (Ia), (II), (IIa), (IIb), (IIIc)and/or (IIIb) according to the invention as defined above show an evenbetter stimulation of the innate immune system, when prepared by an invitro transcription due to its 5′-phosphate, when compared to nucleicacid molecules of either formula (I), (Ia), (II), (IIa), (IIb), (IIIc)and/or (IIIb) according to the invention prepared by synthetic methods.Such a stimulation of the innate immune system is, without being boundthereto, contributed to the activation of the receptor RIG-1.Accordingly, nucleic acid molecules of either formula (I), (Ia), (II),(IIa), (IIb), (IIIc) and/or (IIIb) according to the invention as definedabove are particularly preferred, when prepared by an in vitrotranscription reaction.

The nucleic acid molecule of either formula (I), (Ia), (II), (IIa),(IIb), (IIIc) and/or (IIIb) according to the invention as defined aboveis typically provided as a “stabilized oligonucleotide”, that is to sayas an oligoribonucleotide or oligodeoxyribonucleotide that is resistantto in vivo degradation (e.g. by an exo- or endo-nuclease). Suchstabilization can be effected, for example, by a modified phosphatebackbone of the nucleic acid molecule of either formula (I), (Ia), (II),(IIa), (IIb), (IIIc) and/or (IIIb) according to the invention as definedabove. Nucleotides that are preferably used in this connection contain aphosphorothioate-modified phosphate backbone, preferably at least one ofthe phosphate oxygens contained in the phosphate backbone being replacedby a sulfur atom. Other stabilized oligonucleotides include, forexample: non-ionic analogues, such as, for example, alkyl and arylphosphonates, in which the charged phosphonate oxygen is replaced by analkyl or aryl group, or phosphodiesters and alkylphosphotriesters, inwhich the charged oxygen residue is present in alkylated form. However,the naturally occurring phosphodiester backbone is still preferred.

The nucleic acid molecule of either formula (I), (Ia), (II), (IIa),(IIb), (IIIc) and/or (IIIb) according to the invention as defined abovecan likewise be stabilized. As mentioned above, any nucleic acid, forexample DNA or RNA, can in principle be used for the nucleic acidmolecule of either formula (I), (Ia), (II), (IIa), (IIb), (IIIc) and/or(IIIb) according to the invention as defined above. From the point ofview of safety, however, the use of RNA for such a nucleic acid moleculeis preferred. In particular, RNA does not involve the risk of beingstably integrated into the genome of the transfected cell. In addition,RNA is degraded substantially more easily in vivo. Likewise, no anti-RNAantibodies have hitherto been detected, presumably owing to therelatively short half-life of RNA in vivo as compared with DNA. Incomparison with DNA, RNA is considerably less stable in solution, whichis, inter alia, due substantially to RNA-degrading enzymes, so-calledRNases (ribonucleases). Even the smallest ribonuclease contaminationsare sufficient to degrade RNA completely in solution. Such RNasecontaminations can generally be removed only by special treatment, inparticular with diethyl pyrocarbonate (DEPC). Accordingly, the naturaldegradation of mRNA in the cytoplasm of cells is very finely regulated.A number of mechanisms are known in this connection in the prior art.Thus, the terminal structure is typically of critical importance for anmRNA in vivo. At the 5′ end of naturally occurring mRNAs there isusually a so-called “cap structure” (a modified guanosine (guanine)nucleotide) and at the 3′ end a sequence of up to 200 adenosine(adenine) nucleotides (the so-called poly-A tail).

The nucleic acid molecule of either formula (I), (Ia), (II), (IIa),(IIb), (IIIc) and/or (IIIb) according to the invention as defined above,particularly if provided as an (m)RNA, can therefore be stabilizedagainst degradation by RNases by the addition of a so-called “5′ Cap”structure. Particular preference is given in this connection to am7G(5′)ppp (5′(A,G(5′)ppp(5′)A or G(5′)ppp(5′)G as the 5′ Cap”structure. However, such a modification is introduced only if amodification, for example a lipid modification, has not already beenintroduced at the 5′ end of the nucleic acid molecule of either formula(I), (Ia), (II), (IIa), (IIb), (IIIc) and/or (IIIb) according to theinvention as defined above or if the modification does not interferewith the immunogenic properties of the (unmodified or chemicallymodified) nucleic acid molecule of either formula (I), (Ia), (II),(IIa), (IIb), (IIIc) and/or (IIIb) according to the invention as definedabove.

Alternatively, the 3′ end of the nucleic acid molecule of either formula(I), (Ia), (II), (IIa), (IIb), (IIIc) and/or (IIIb) according to theinvention as defined above, particularly if provided as an RNA, can bemodified by a sequence of at least 50 adenosine ribonucleotides,preferably at least 70 adenosine ribonucleotides, more preferably atleast 100 adenosine ribonucleotides, particularly preferably at least200 adenosine (adenine) ribonucleotides (so-called “poly-A tail”).Particularly, the nucleic acid molecule of either formula (I), (Ia),(II), (IIa), (IIb), (IIIc) and/or (IIIb) according to the invention asdefined above may contain, especially if the RNA is in the form of an(m)RNA, a poly-A tail on the 3′ terminus of typically about 10 to 200adenosine nucleotides, preferably about 10 to 100 adenosine nucleotides,more preferably about 20 to 100 adenosine nucleotides or even morepreferably about 40 to 80 adenosine nucleotides.

Furthermore, the 3′ end of the nucleic acid molecule of either formula(I), (Ia), (II), (IIa), (IIb), (IIIc) and/or (IIIb) according to theinvention as defined above, particularly if provided as an RNA, can bemodified by a sequence of at least 50 cytidine ribonucleotides,preferably at least 70 cytidine ribonucleotides, more preferably atleast 100 cytidine ribonucleotides, particularly preferably at least 200cytidine ribonucleotides (so-called “poly-C tail”). Particularly, thenucleic acid molecule of either formula (I), (Ia), (II), (IIa), (IIb),(IIIc) and/or (IIIb) according to the invention as defined above maycontain, especially if the RNA is in the form of an (m)RNA, a poly-Ctail on the 3′ terminus of typically about 10 to 200 cytidinenucleotides, preferably about 10 to 100 cytidine nucleotides, morepreferably about 20 to 70 cytidine nucleotides or even more preferablyabout 20 to 60 or even 10 to 40 cytidine nucleotides.

Analogously, in this case too, such a (“poly-A tail” and/or “poly-Ctail”-) modification can be introduced only if no modification, forexample a lipid modification, has already been introduced at the 3′ endof the nucleic acid molecule of either formula (I), (Ia), (II), (IIa),(IIb), (IIIc) and/or (IIIb) according to the invention as defined aboveor if the modification does not interfere with the immunogenicproperties of the (unmodified or chemically modified) nucleic acidmolecule of either formula (I), (Ia), (II), (IIa), (IIb), (IIIc) and/or(IIIb) according to the invention as defined above.

The above-mentioned modifications, that is to say the insertion of a “5′Cap” structure or the insertion of a “poly-A tail” and/or a “poly-Ctail” at the 3′ end, prevent premature degradation of the nucleic acidmolecule of either formula (I), (Ia), (II), (IIa), (IIb), (IIIc) and/or(IIIb) according to the invention as defined above in vivo andaccordingly stabilize the nucleic acid molecule of either formula (I),(Ia), (II), (IIa), (IIb), (IIIc) and/or (IIIb) according to theinvention as defined above in vivo.

According to a particular embodiment, the nucleic acid molecule ofeither formula (I), (Ia), (II), (IIa), (IIb), (IIIc) and/or (IIIb)according to the invention as defined above can contain a lipidmodification. Such a lipid-modified nucleic acid molecule according tothe invention typically comprises a nucleic acid molecule of eitherformula (I), (Ia), (II), (IIa), (IIb), (IIIc) and/or (IIIb) according tothe invention as defined above, at least one linker covalently linkedwith that nucleic acid molecule according to the invention, and at leastone lipid covalently linked with the respective linker. Alternatively,the lipid-modified nucleic acid molecule according to the inventioncomprises a (at least one) nucleic acid molecule of either formula (I),(Ia), (II), (IIa), (IIb), (IIIc) and/or (IIIb) according to theinvention as defined above and at least one (bifunctional) lipidcovalently linked (without a linker) with that nucleic acid moleculeaccording to the invention. According to a third alternative, thelipid-modified nucleic acid molecule according to the inventioncomprises a nucleic acid molecule of either formula (I), (Ia), (II),(IIa), (IIb), (IIIc) and/or (IIIb) according to the invention as definedabove, at least one linker covalently linked with that nucleic acidmolecule according to the invention, and at least one lipid covalentlylinked with the respective linker, and also at least one (bifunctional)lipid covalently linked (without a linker) with that nucleic acidmolecule according to the invention.

The lipid contained in the lipid-modified nucleic acid moleculeaccording to the invention is typically a lipid or a lipophilic residuethat preferably is itself biologically active. Such lipids preferablyinclude natural substances or compounds such as, for example, vitamins,e.g. α-tocopherol (vitamin E), including RRR-α-tocopherol (formerlyD-α-tocopherol), L-α-tocopherol, the racemate D,L-α-tocopherol, vitaminE succinate (VES), or vitamin A and its derivatives, e.g. retinoic acid,retinol, vitamin D and its derivatives, e.g. vitamin D and also theergosterol precursors thereof, vitamin E and its derivatives, vitamin Kand its derivatives, e.g. vitamin K and related quinone or phytolcompounds, or steroids, such as bile acids, for example cholic acid,deoxycholic acid, dehydrocholic acid, cortisone, digoxygenin,testosterone, cholesterol or thiocholesterol. Further lipids orlipophilic residues within the scope of the present invention include,without implying any limitation, polyalkylene glycols (Oberhauser etal., Nucl. Acids Res., 1992, 20, 533), aliphatic groups such as, forexample, C₁-C₂₀-alkanes, C₁-C₂₀-alkenes or C₁-C₂₀-alkanol compounds,etc., such as, for example, dodecanediol, hexadecanol or undecylresidues (Saison-Behmoaras et al., EMBO J, 1991, 10, 111; Kabanov etal., FEBS Lett., 1990, 259, 327; Svinarchuk et al., Biochimie, 1993, 75,49), phospholipids such as, for example, phosphatidylglycerol,diacylphosphatidylglycerol, phosphatidylcholine,dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, di-hexadecyl-rac-glycerol,sphingolipids, cerebrosides, gangliosides, or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl. Acids Res., 1990,18, 3777), polyamines or polyalkylene glycols, such as, for example,polyethylene glycol (PEG) (Manoharan et al., Nucleosides & Nucleotides,1995, 14, 969), hexaethylene glycol (HEG), palmitin or palmityl residues(Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229),octadecylamines or hexylamino-carbonyl-oxycholesterol residues (Crookeet al., J. Pharmacol. Exp. Ther., 1996, 277, 923), and also waxes,terpenes, alicyclic hydrocarbons, saturated and mono- orpoly-unsaturated fatty acid residues, etc.

Linking between the lipid and the nucleic acid molecule of eitherformula (I), (Ia), (II), (IIa), (IIb), (IIIc) and/or (IIIb) according tothe invention as defined above can in principle take place at anynucleotide, at the base or the sugar component of any nucleotide of theinventive nucleic acid, at the 3′ and/or 5′ end, and/or at the phosphatebackbone of the nucleic acid molecule of either formula (I), (Ia), (II),(IIa), (IIb), (IIIc) and/or (IIIb) according to the invention as definedabove. Particular preference is given according to the invention to aterminal lipid modification of the nucleic acid molecule according tothe invention at the 3′ and/or 5′ end thereof. A terminal modificationhas a number of advantages over modifications within the sequence. Onthe one hand, modifications within the sequence can influence thehybridisation behaviour, which may have an adverse effect in the case ofsterically demanding residues. On the other hand, in the case of thesynthetic preparation of a lipid-modified nucleic acid moleculeaccording to the invention that is modified only terminally, thesynthesis of the nucleic acid molecule of either formula (I), (Ia),(II), (IIa), (IIb), (IIIc) and/or (IIIb) according to the invention asdefined above can be carried out with commercially available monomersthat are obtainable in large quantities, and synthesis protocols knownin the prior art can be used.

According to a first preferred embodiment, linking between the nucleicacid molecule according to the invention and at least one lipid that isused is effected via a “linker” (covalently linked with the nucleic acidmolecule of either formula (I), (Ia), (II), (IIa), (IIb), (IIIc) and/or(IIIb) according to the invention as defined above). Linkers within thescope of the present invention typically have at least two andoptionally 3, 4, 5, 6, 7, 8, 9, 10, 10-20, 20-30 or more reactivegroups, selected from, for example, a hydroxy group, an amino group, analkoxy group, etc. One reactive group preferably serves to bind theabove-described nucleic acid molecule of either formula (I), (Ia), (II),(IIa), (IIb), (IIIc) and/or (IIIb) according to the invention as definedabove, for example an RNA oligonucleotide. This reactive group can bepresent in protected form, for example as a DMT group (dimethoxytritylchloride), as a Fmoc group, as a MMT (monomethoxytrityl) group, as a TFA(trifluoroacetic acid) group, etc. Furthermore, sulfur groups can beprotected by disulfides, for example alkylthiols such as, for example,3-thiopropanol, or by activated components such as 2-thiopyridine. Oneor more further reactive groups serve according to the invention for thecovalent binding of one or more lipids. According to the firstembodiment, therefore, a nucleic acid molecule of either formula (I),(Ia), (II), (IIa), (IIb), (IIIc) and/or (IIIb) according to theinvention as defined above can bind via the covalently bound linkerpreferably at least one lipid, for example 1, 2, 3, 4, 5, 5-10, 10-20,20-30 or more lipid(s), particularly preferably at least 3-8 or morelipid(s) per nucleic acid molecule of either formula (I), (Ia), (II),(IIa), (IIb), (IIIc) and/or (IIIb) according to the invention as definedabove. The bound lipids can thereby be bound separately from one anotherat different positions of the nucleic acid molecule of either formula(I), (Ia), (II), (IIa), (IIb), (IIIc) and/or (IIIb) according to theinvention as defined above, or they can be present in the form of acomplex at one or more positions of the nucleic acid molecule of eitherformula (I), (Ia), (II), (IIa), (IIb), (IIIc) and/or (IIIb) according tothe invention as defined above. An additional reactive group of thelinker can be used for direct or indirect (cleavable) binding to acarrier material, for example a solid phase. Preferred linkers accordingto the present invention are, for example, glycol, glycerol and glycerolderivatives, 2-aminobutyl-1,3-propanediol and2-aminobutyl-1,3-propanediol derivatives/skeleton, pyrrolidine linkersor pyrrolidine-containing organic molecules (in particular for amodification at the 3′ end), etc. Glycerol or glycerol derivatives (C₅anchor) or a 2-aminobutyl-1,3-propanediol derivative/skeleton (C₇anchor) are particularly preferably used according to the invention aslinkers. A glycerol derivative (C₅ anchor) as linker is particularlypreferred when the lipid modification can be introduced via an etherbond. If the lipid modification is to be introduced via an amide or aurethane bond, for example, a 2-aminobutyl-1,3-propanediol skeleton (C₇anchor), for example, is preferred. In this connection, the nature ofthe bond formed between the linker and the nucleic acid molecule ofeither formula (I), (Ia), (II), (IIa), (IIb), (IIIc) and/or (IIIb)according to the invention as defined above is preferably such that itis compatible with the conditions and chemicals of amidite chemistry,that is to say it is preferably neither acid- nor base-labile.Preference is given in particular to bonds that are readily obtainablesynthetically and are not hydrolysed by the ammoniacal cleavageprocedure of a nucleic acid synthesis process. Suitable bonds are inprinciple all correspondingly suitable bonds, preferably ester bonds,amide bonds, urethane and ether bonds. In addition to the goodaccessibility of the starting materials (few synthesis steps),particular preference is given to the ether bond owing to its relativelyhigh biological stability towards enzymatic hydrolysis.

According to a second preferred embodiment, the (at least one) nucleicacid molecule of either formula (I), (Ia), (II), (IIa), (IIb), (IIIc)and/or (IIIb) according to the invention as defined above is linkeddirectly with at least one (bifunctional) lipid as described above, thatis to say without the use of a linker as described above. In this case,the (bifunctional) lipid used according to the invention preferablycontains at least two reactive groups or optionally 3, 4, 5, 6, 7, 8, 9,10 or more reactive groups, a first reactive group serving to bind thelipid directly or indirectly to a carrier material described herein andat least one further reactive group serving to bind a nucleic acidmolecule of either formula (I), (Ia), (II), (IIa), (IIb), (IIIc) and/or(IIIb) according to the invention as defined above. According to thesecond embodiment, a nucleic acid molecule of either formula (I), (Ia),(II), (IIa), (IIb), (IIIc) and/or (IIIb) according to the invention asdefined above can therefore preferably bind at least one lipid (directlywithout a linker), for example 1, 2, 3, 4, 5, 5-10, 10-20, 20-30 or morelipid(s), particularly preferably at least 3-8 or more lipid(s) pernucleic acid molecule of either formula (I), (Ia), (II), (IIa), (IIb),(IIIc) and/or (IIIb) according to the invention as defined above. Thebound lipids can be bound separately from one another at differentpositions of the nucleic acid molecule of either formula (I), (Ia),(II), (IIa), (IIb), (IIIc) and/or (IIIb) according to the invention asdefined above, or they can be present in the form of a complex at one ormore positions of the nucleic acid molecule of either formula (I), (Ia),(II), (IIa), (IIb), (IIIc) and/or (IIIb) according to the invention asdefined above. Alternatively, at least one nucleic acid molecule ofeither formula (I), (Ia), (II), (IIa), (IIb), (IIIc) and/or (IIIb)according to the invention as defined above, for example optionally 3,4, 5, 6, 7, 8, 9, 10, 10-20, 20-30 or more nucleic acids of eitherformula (I), (Ia), (II), (IIa), (IIb), (IIIc) and/or (IIIb) according tothe invention as defined above, can be bound according to the secondembodiment to a lipid as described above via its reactive groups. Lipidsthat can be used for this second embodiment particularly preferablyinclude those (bifunctional) lipids that permit coupling (preferably attheir termini or optionally intramolecularly), such as, for example,polyethylene glycol (PEG) and derivatives thereof, hexaethylene glycol(HEG) and derivatives thereof, alkanediols, aminoalkane, thioalkanols,etc. The nature of the bond between a (bifunctional) lipid and a nucleicacid molecule of either formula (I), (Ia), (II), (IIa), (IIb), (IIIc)and/or (IIIb) according to the invention as defined above, as describedabove, is preferably as described for the first preferred embodiment.

According to a third embodiment, linking between the nucleic acidmolecule of either formula (I), (Ia), (II), (IIa), (IIb), (IIIc) and/or(IIIb) according to the invention as defined above and at least onelipid as described above can take place via both of the above-mentionedembodiments simultaneously. For example, the nucleic acid molecule ofeither formula (I), (Ia), (II), (IIa), (IIb), (IIIc) and/or (IIIb)according to the invention as defined above can be linked at oneposition of the nucleic acid with at least one lipid via a linker(analogously to the first embodiment) and at a different position of thenucleic acid molecule of either formula (I), (Ia), (II), (IIa), (IIb),(IIIc) and/or (IIIb) according to the invention as defined abovedirectly with at least one lipid without the use of a linker(analogously to the second embodiment). For example, at the 3′ end of anucleic acid molecule of either formula (I), (Ia), (II), (IIa), (IIb),(IIIc) and/or (IIIb) according to the invention as defined above, atleast one lipid as described above can be covalently linked with thenucleic acid via a linker, and at the 5′ end of the nucleic acidmolecule according to the invention, a lipid as described above can becovalently linked with the nucleic acid without a linker. Alternatively,at the 5′ end of a nucleic acid molecule of either formula (I), (Ia),(II), (IIa), (IIb), (IIIc) and/or (IIIb) according to the invention asdefined above, at least one lipid as described above can be covalentlylinked with the nucleic acid molecule via a linker, and at the 3′ end ofthe nucleic acid molecule of either formula (I), (Ia), (II), (IIa),(IIb), (IIIc) and/or (IIIb) according to the invention as defined above,a lipid as described above can be covalently linked with the nucleicacid molecule without a linker. Likewise, covalent linking can takeplace not only at the termini of the nucleic acid molecule of eitherformula (I), (Ia), (II), (IIa), (IIb), (IIIc) and/or (IIIb) according tothe invention as defined above but also intramolecularly, as describedabove, for example at the 3′ end and intramolecularly, at the 5′ end andintramolecularly, at the 3′ and 5′ end and intramolecularly, onlyintramolecularly, etc.

The lipid-modified nucleic acid molecule of either formula (I), (Ia),(II), (IIa), (IIb), (IIIc) and/or (IIIb) according to the invention asdefined above can preferably be obtained by various processes. The lipidmodification can in principle—as defined above—be introduced at anyposition of the nucleic acid molecule of either formula (I), (Ia), (II),(IIa), (IIb), (IIIc) and/or (IIIb) according to the invention as definedabove, for example at the 3′ and/or 5′ ends or at the phosphate backboneof the nucleic acid molecule of either formula (I), (Ia), (II), (IIa),(IIb), (IIIc) and/or (IIIb) according to the invention as defined aboveand/or at any base or at the sugar of any nucleotide of the nucleic acidmolecule of either formula (I), (Ia), (II), (IIa), (IIb), (IIIc) and/or(IIIb) according to the invention as defined above. According to theinvention, preference is given to terminal lipid modifications at the 3′and/or 5′ ends of the nucleic acids of either formula (I), (Ia), (II),(IIa), (IIb), (IIIc) and/or (IIIb) according to the invention as definedabove. By means of such a terminal chemical modification it is possibleaccording to the invention to obtain a large number of differentlyderivatised nucleic acids. The process for preparing such lipid-modifiednucleic acids of either formula (I), (Ia), (II), (IIa), (IIb), (IIIc)and/or (IIIb) according to the invention as defined above is preferablychosen in dependence on the position of the lipid modification.

If, for example, the lipid modification takes place at the 3′ end of thenucleic acid molecule of either formula (I), (Ia), (II), (IIa), (IIb),(IIIc) and/or (IIIb) according to the invention as defined above, thenthe lipid modification is typically carried out either before or afterthe preparation of the nucleic acid molecule of either formula (I),(Ia), (II), (IIa), (IIb), (IIIc) and/or (IIIb) according to theinvention as defined above. The preparation of the nucleic acid moleculeof either formula (I), (Ia), (II), (IIa), (IIb), (IIIc) and/or (IIIb)according to the invention as defined above can be carried out by directsynthesis of the nucleic acid or optionally by addition of a readysynthesized nucleic acid or a nucleic acid from samples isolated fromother sources.

According to a first alternative, the nucleic acid molecule of eitherformula (I), (Ia), (II), (IIa), (IIb), (IIIc) and/or (IIIb) according tothe invention as defined above is synthesized directly beforeintroduction of the lipid, typically by means of processes known in theprior art for the synthesis of nucleic acids. To this end, a startingnucleotide (nucleoside) is preferably bound to a solid phase, forexample via a coupling molecule, e.g. a succinyl residue, and thenucleic acid molecule of either formula (I), (Ia), (II), (IIa), (IIb),(IIIc) and/or (IIIb) according to the invention as defined above issynthesized, for example by the process of amidite chemistry. A linkeras described hereinbefore is then covalently bonded, preferably via afirst reactive group of the linker, to the 3′ end of the nucleic acidmolecule of either formula (I), (Ia), (II), (IIa), (IIb), (IIIc) and/or(IIIb) according to the invention as defined above. A lipid as describedhereinbefore can then be covalently linked with the linker via a secondreactive group of the linker. Alternatively, the linker can becovalently linked with the lipid before it is bound to the 3′ end of thenucleic acid molecule of either formula (I), (Ia), (II), (IIa), (IIb),(IIIc) and/or (IIIb) according to the invention as defined above. Inthis case, only the binding of a first reactive group of the linker withthe 3′ end of the nucleic acid molecule of either formula (I), (Ia),(II), (IIa), (IIb), (IIIc) and/or (IIIb) according to the invention asdefined above is necessary. After synthesis of the nucleic acid moleculeof either formula (I), (Ia), (II), (IIa), (IIb), (IIIc) and/or (IIIb)according to the invention as defined above, or after binding of thelipid, the nucleic acid molecule of either formula (I), (Ia), (II),(IIa), (IIb), (IIIc) and/or (IIIb) according to the invention as definedabove can be separated from the solid phase and deprotected. If thesynthesis has been carried out in solution, a washing and purificationstep for removing unreacted reactants as well as solvents andundesirable secondary products can be carried out after the synthesis ofthe lipid-modified nucleic acid molecule according to the invention (andoptionally before separation from the carrier material).

According to a further alternative, a 3′-lipid-modified nucleic acidmolecule of either formula (I), (Ia), (II), (IIa), (IIb), (IIIc) and/or(IIIb) according to the invention as defined above, as defined above, issynthesized after introduction of the lipid on a reactive group of thelinker or is bound to the reactive group of the linker as a readysynthesized nucleic acid molecule of either formula (I), (Ia), (II),(IIa), (IIb), (IIIc) and/or (IIIb) according to the invention as definedabove. To this end, for example, a first reactive group of a linker asdescribed above can be reacted with a lipid as described hereinbefore.Then, preferably in a second step, a second reactive group of the linkeris provided with an acid-stable protecting group, e.g. DMT, Fmoc, etc.,in order to permit subsequent binding of the nucleic acid molecule ofeither formula (I), (Ia), (II), (IIa), (IIb), (IIIc) and/or (IIIb)according to the invention as defined above to that reactive group. Thelinker can then be bound directly or indirectly to a solid phase via athird reactive group of the linker. Indirect binding is possible, forexample, via a (coupling) molecule, which can be bound both covalentlyto the linker and to the solid phase. Such a (coupling) molecule is, forexample, a succinyl residue, etc., as described hereinbelow. Removal ofthe protecting group at the third reactive group of the linker and thebinding or synthesis of the nucleic acid molecule of either formula (I),(Ia), (II), (IIa), (IIb), (IIIc) and/or (IIIb) according to theinvention as defined above at the reactive group that is now accessiblethen usually take place. Finally, the lipid-modified nucleic acidmolecule according to the invention is typically cleaved from thecarrier material (and the protective groups on the nucleic acid areoptionally removed). However, a further lipid can optionally also becoupled to the 3′ end of the coupled nucleic acid molecule according tothe invention of either formula (I), (Ia), (II), (IIa), (IIb), (IIIc)and/or (IIIb), preferably according to one of the steps describedhereinbefore.

According to a variant of this above-mentioned alternative, a linker asdescribed above can be bound directly or indirectly to a solid phase viaa first reactive group. An acid-stable protecting group is then firstbound to a second reactive group of the linker. After binding of theprotecting group to the second reactive group, a lipid as describedabove can first be bound to a third reactive group of the linker. Thenthere are likewise preferably carried out the removal of the protectinggroup at the third reactive group of the linker, the binding orsynthesis of a nucleic acid molecule of either formula (I), (Ia), (II),(IIa), (IIb), (IIIc) and/or (IIIb) according to the invention as definedabove at the reactive group that is now accessible, and the cleavage ofthe lipid-modified nucleic acid molecule according to the invention fromthe carrier material (and optionally the removal of the protectinggroups at the nucleic acid).

According to a particularly preferred embodiment of the 3′-lipidmodification of a nucleic acid molecule of either formula (I), (Ia),(II), (IIa), (IIb), (IIIc) and/or (IIIb) according to the invention asdefined above, such a lipid-modified nucleic acid molecule according tothe invention can be synthesized via a linker having three reactivegroups (a trifunctional anchor compound) based on a glycerol fundamentalsubstance (C₅ anchor) and having a monofunctional lipid, such as, forexample, a palmityl residue, cholesterol or tocopherol. As startingmaterial for the synthesis of the linker there can be used, for example,alpha,beta-isopropylidene-glycerol (a glycerol containing a ketalprotecting group), which is preferably first converted into thealcoholate with sodium hydride and is reacted with hexadecyl bromide anda lipid in a Williamson synthesis to form the corresponding ether.Alternatively, the ether bond can be linked in the first step by adifferent method, for example by formation of a tosylate ofα,β-isopropylidene-glycerol, and reaction of the tosylate with thereactive group of a lipid, for example an acidic proton, to form thecorresponding ether. In a second stage, the ketal protecting group canbe removed with an acid, for example acetic acid, dilute hydrochloricacid, etc., and then the primary hydroxy group of the diol can beprotected selectively by dimethoxytrityl chloride (DMT-Cl). In the laststage, the reaction of the product obtained in the preceding step withsuccinic anhydride is preferably carried out to form the succinate withDMAP as catalyst. Such a linker is particularly suitable, for example,for the binding of palmityl residues or tocopherol as lipid.

According to another alternative, the 3′-lipid modification of a nucleicacid molecule of either formula (I), (Ia), (II), (IIa), (IIb), (IIIc)and/or (IIIb) according to the invention as defined above, is effectedusing a (bifunctional) lipid, such as, for example, polyethylene glycol(PEG) or hexaethylene glycol (HEG), without using a linker as describedabove. Such bifunctional lipids typically have two functional groups asdescribed above, wherein one end of the bifunctional lipid canpreferably be bound to the carrier material via a (coupling) molecule,for example a base-labile succinyl anchor, etc., as described herein,and the nucleic acid molecule of either formula (I), (Ia), (II), (IIa),(IIb), (IIIc) and/or (IIIb) according to the invention as defined abovecan be synthesized at the other end of the bifunctional lipid (E. Bayer,M. Maier, K. Bleicher, H.-J. Gaus Z. Naturforsch. 50b (1995) 671). Bythe omission of the third functionalisation and of a linker,respectively, as used hereinbefore, the synthesis of such alipid-modified nucleic acid molecule according to the invention issimplified. For the preparation, the bifunctional lipid used accordingto the invention, for example polyethylene glycol, is typically firstmonosubstituted with a protecting group, for example DMT. In a secondstage, esterification of the lipid protected at a reactive group isusually carried out with succinic anhydride, with DMAP catalysis, toform the succinate. Thereafter, in a third stage, the bifunctional lipidcan be coupled to a carrier material and deprotected, following whichthe synthesis of the nucleic acid molecule of either formula (I), (Ia),(II), (IIa), (IIb), (IIIc) and/or (IIIb) according to the invention asdefined above takes place in a fourth step in accordance with a processas described hereinbefore. Deprotection of the synthesized nucleic acidmolecule of either formula (I), (Ia), (II), (IIa), (IIb), (IIIc) and/or(IIIb) according to the invention as defined above and cleavage of thelipid-modified nucleic acid from the carrier material are thenoptionally carried out.

According to another preferred embodiment, the lipid modification of anucleic acid molecule of either formula (I), (Ia), (II), (IIa), (IIb),(IIIc) and/or (IIIb) according to the invention as defined above, takesplace at the 5′ end of the nucleic acid. The lipid modification isthereby typically carried out either after the provision or after thesynthesis of the nucleic acid molecule of either formula (I), (Ia),(II), (IIa), (IIb), (IIIc) and/or (IIIb) according to the invention asdefined above. The provision of the nucleic acid molecule of eitherformula (I), (Ia), (II), (IIa), (IIb), (IIIc) and/or (IIIb) according tothe invention as defined above can be carried out—as defined above—via adirect synthesis of the nucleic acid molecule of either formula (I),(Ia), (II), (IIa), (IIb), (IIIc) and/or (IIIb) according to theinvention as defined above or by addition of a ready synthesized nucleicacid molecule of either formula (I), (Ia), (II), (IIa), (IIb), (IIIc)and/or (IIIb) according to the invention as defined above. A synthesisof the nucleic acid molecule of either formula (I), (Ia), (II), (IIa),(IIb), (IIIc) and/or (IIIb) according to the invention as defined abovetakes place, preferably analogously to the method described above,according to processes of nucleic acid synthesis known in the prior art,more preferably according to the phosphoramidite process.

According to a particularly preferred embodiment, the lipid modificationof a nucleic acid molecule of either formula (I), (Ia), (II), (IIa),(IIb), (IIIc) and/or (IIIb) according to the invention as defined abovetakes place at the 5′ end of the nucleic acid molecule according to theinvention by specially modified phosphoramidites following aphosphoramidite process for the synthesis of the nucleic acid. Suchamidites, which are obtainable quite simply by synthesis, areconventionally coupled as the last monomer to a commercially availableor to a ready synthesized nucleic acid. These reactions aredistinguished by a rather rapid reaction kinetics and very high couplingyields. The synthesis of the modified amidites preferably takes place byreaction of a phosphoramidite, for examplebeta-cyanoethyl-monochlorophosphoramidite (phosphorous acidmono-(2-cyanoethyl ester)-diisopropyl-amide chloride), with an alcohol,dissolved in a suitable solvent, for example in absolutedichloromethane, of a lipid as defined above, for example a lipidalcohol of tocopherol, cholesterol, hexadecanol, DMT-PEG, etc. Likewisepreferably, DIPEA is added to the reaction solution as acid acceptor.

These phosphoramidites used for the synthesis of the 5′-lipid-modifiednucleic acids according to the invention are relatively resistant tohydrolysis and can (prior to the synthesis) be purifiedchromatographically by means of silica gel. To this end, a small amountof a weak base, such as, for example, triethylamine, is typically addedto the eluent in order to avoid decomposition of the amidite. It isimportant that this base is removed completely from the product again,in order to avoid poor coupling yields. This can be carried out, forexample, by simple drying in vacuo, but preferably by purification ofthe phosphoramidites by precipitation thereof from tert-butyl methylether using pentane. If the lipid-modified amidites used have a veryhigh viscosity, for example are present in the form of a viscous oil,(rapid) column chromatography can also be carried out, which makes itpossible to dispense with triethylamine as base. Such a purification istypically not carried out in the case of PEG-modified amidites, however,because they contain the acid-labile DMT protecting group.

For the coupling reaction of the lipid-modified phosphoramidites to the5′ end of a nucleic acid molecule of either formula (I), (Ia), (II),(IIa), (IIb), (IIIc) and/or (IIIb) according to the invention as definedabove there are preferably used those solvents in which the amiditesused are sufficiently soluble. For example, owing to the highlipophilicity of the amidites used according to the invention, theirsolubility in acetonitrile can be limited. Apart from acetonitrile asthe solvent that is typically used, a solution of chlorinatedhydrocarbons is therefore preferably used for the coupling reactions,for example a 0.1 M solution in (absolute) dichloromethane. The use ofdichloromethane requires some changes to the standard protocol of thesynthesis cycle, however. For example, in order to avoid precipitationof the amidite in the pipes of the automatic synthesis device and on thecarrier material, all the valves and pipes that come into contact withthe amidite are flushed with (absolute) dichloromethane before and afterthe actual coupling step and blown dry.

When lipid-modified amidites are used, high coupling yields aretypically obtained, which are comparable with the coupling yield ofamidites conventionally used in the prior art. The kinetics of thereaction of lipid-modified amidites generally proceeds more slowly. Forthis reason, the coupling times are preferably (markedly) lengthenedwhen lipid-modified amidites are used, as compared with standardprotocols. Such coupling times can easily be determined by a personskilled in the art. Because a capping step after the coupling can beomitted, it is likewise possible, if required, to carry out a furthersynthesis cycle with the same lipid-modified amidite, in order toincrease the overall yield of the reaction. In this case, thedetritylation step is not usually carried out, for example in the caseof DMT-modified lipids such as DMT-PEG.

In the synthesis of 5′-lipid-modified nucleic acid molecules accordingto the invention, the phosphite triester via which the lipid is bound tothe nucleic acid molecule of either formula (I), (Ia), (II), (IIa),(IIb), (IIIc) and/or (IIIb) according to the invention as defined abovecan be oxidised by a sulfurising agent. To this end there is preferablyused a sulfurising agent that achieves oxidation of the phosphotriesteras completely as possible. Otherwise, the sulfurisation reaction, forexample for steric reasons, may proceed so incompletely that only asmall amount of product, or no product at all, is obtained after theammoniacal cleavage and deprotection of the MON. This phenomenon isdependent on the type of modification, the sulfurising agent used andthe sulfurisation conditions. The oxidation is therefore carried outpreferably with iodine. As a result, although a phosphodiester bond isintroduced, it is not to be expected, owing to the proximity of thelipid residue, that this bond will be recognised as a substrate bynucleases.

In a lipid modification, linkers or (bifunctional) lipids contained inthe nucleic acid molecule of either formula (I), (Ia), (II), (IIa),(IIb), (IIIc) and/or (IIIb) according to the invention as defined above,or optionally the nucleic acid molecule of either formula (I), (Ia),(II), (IIa), (IIb), (IIIc) and/or (IIIb) according to the invention asdefined above itself, can, as described hereinbefore, be coupleddirectly or indirectly to a carrier material. Direct coupling is carriedout preferably directly with the carrier material, while indirectcoupling to the carrier material is typically carried out via a further(coupling) molecule. The bond formed by the coupling to a carriermaterial preferably exhibits a (cleavable) covalent bond with the linkeror bifunctional lipid and/or a (cleavable) covalent bond with the solidphase. Compounds suitable as (coupling) molecule are, for example,dicarboxylic acids, for example succinyl residues (=succinyl anchors),oxalyl residues (=oxalyl anchors), etc. Linkers, (bifunctional) lipidsor optionally nucleic acids of either formula (I), (Ia), (II), (IIa),(IIb), (IIIc) and/or (IIIb) according to the invention as defined abovewhich, like, for example, aminoalkyl residues (e.g. aminopropyl oraminohexanyl residues), carry a free amino function, can be bound to thecarrier material via a phthalimide linker. Thiol-containing linkers,(bifunctional) lipids or optionally nucleic acids of either formula (I),(Ia), (II), (IIa), (IIb), (IIIc) and/or (IIIb) according to theinvention as defined above can be bound in disulfide form to the carriermaterial. Suitable carrier materials in connection with this inventionare in particular solid phases such as CPG, Tentagel®,amino-functionalised PS-PEG (Tentagel® S NH₂), etc., preferablyTentagel® or amino-functionalised PS-PEG (Tentagel® S NH₂). According toa particular embodiment it is possible for the coupling to a carriermaterial to couple, for example, the succinates of the described linkersor bifunctional lipids used according to the invention, preferably withTBTU/NMM (1H-benzotriazol-1-yl-1,1,3,3-tetramethyluroniumtetrafluoroborate/N-methylmorpholine) as coupling reagent, toamino-functionalised PS-PEG (Tentagel® S NH₂). In the case of PS-PEGcarrier materials on the 1 μmol scale that is conventionally used, thebest results are typically obtained with loads of from 50 to 100 μmol/g(E. Bayer, K. Bleicher, M. Maier Z. Naturforsch. 50b (1995) 1096). If,however, nucleotides are to be synthesized on a large scale according tothe invention, the loading of the carrier materials is preferably ashigh as possible (≧100 μmol). According to the invention, such a processlikewise results in good coupling yields (M. Gerster, M. Maier, N.Clausen, J. Schewitz, E. Bayer Z. Naturforsch. 52b (1997) 110). Forexample, carrier materials such as, for example, resins with a load ofup to 138 μmol/g or optionally more can be used with good synthesisyields. Because the coupling yields with the above-described linkers orbifunctional lipids are approximately 100%, the loading of the carriermaterial can be adjusted relatively precisely via the stoichiometry ofthese compounds. The loading is preferably monitored by spectroscopicquantification of the cleaved DMT protecting group (see experimentalpart). The residual amino functions still present on the carriermaterial can be capped with acetic anhydride. This capping is normallycarried out following the loading of the carrier material but can alsotake place directly in the nucleic acid synthesis, for example in a DNAsynthesizer. For the synthesis of lipid-modified nucleic acids on thederivatised PS-PEG carrier materials there are preferably used synthesiscycles developed specifically for Tentagel®, which take into account thecharacteristic properties of the material (E. Bayer, M. Maier, K.Bleicher, H.-J. Gaus Z. Naturforsch. Sob (1995) 671, E. Bayer, K.Bleicher, M. Maier Z. Naturforsch. 50b (1995) 1096.). Preferred changesas compared with the standard protocol include:

-   -   lengthened reaction times in the coupling, capping and oxidation        steps;    -   increased number of detritylation steps;    -   lengthened washing steps after each step;    -   use of an ascorbic-acid-containing washing solution (0.1 M in        dioxane/water=9:1) after the oxidation step that is usually        necessary (for oxidation of the phosphite triester) during the        amidite process, in order to remove traces of iodine.

It should be noted that the nature of the modification can have aninfluence on the individual steps of the synthesis cycle. For example,in the case of PEG₁₅₀₀-derivatised carrier materials, a considerablyslowed reaction kinetics is observed, which requires the detritylationsteps to be lengthened again and the coupling time to be lengthened inaddition. Such changes and adaptations are within the scope of thenormal capability of a person skilled in the art and can be carried outat any time within the context of the present disclosure. With thesereaction cycles so modified, both lipid-modified phosphorodiesters andphosphorothioates can be synthesized. The coupling yields of amidites onlinkers or bifunctional lipids used according to the invention are notimpaired by the lipid residues but correspond to conventional values(97-99%). The possibility of 5′ derivatisation and the introduction offurther modifications, for example at base, sugar or phosphate backbone,is retained when such 3′ modifications are used.

The nucleic acid molecule of either formula (I), (Ia), (II), (IIa),(IIb), (IIIc) and/or (IIIb) according to the invention as defined above,as chemically unmodified nucleic acid or as (chemically) modifiednucleic acid, e.g. as a lipid modified nucleic acid molecule of eitherformula (I), (Ia), (II), (IIa), (IIb), (IIIc) and/or (IIIb) according tothe invention as defined above, can likewise be stabilized by forming acomplex of the nucleic acid molecule of either formula (I), (Ia), (II),(IIa), (IIb), (IIIc) and/or (IIIb) according to the invention as definedabove, e.g., without being limited thereto, with a cationic polymer,cationic peptides or polypeptides, preferably with a polycationicpolymer such as polylysine or polyarginine or alternatively withcationic lipids or lipofectants, with a histone, a nucleoline,protamine, oligofectamine, spermine or spermidine, and cationicpolysaccharides, in particular chitosan, TDM, MDP, muramyl dipeptide,pluronics, and/or one of the derivatives thereof, etc. Histones andprotamines are cationic proteins which naturally compact DNA. They arethus responsible in vivo for the condensation of non-transcribed DNA andthe DNA of certain viruses. As histones which may be used in the contextof the present invention to form a complex with the nucleic acidmolecule of either formula (I), (Ia), (II), (IIa), (IIb), (IIIc) and/or(IIIb) according to the invention as defined above, mention may be mademore particularly of histones H1, H2a, H3 and H4. However, protamin(protamin P1 or P2) or cationic partial sequences of protamine arespecifically preferred. In the context of the present invention, thecompound may advantageously be represented by a peptide sequence derivedfrom the protamin P1 or P2, and more precisely corresponding to the(cationic) sequence (SRSRYYRQRQRSRRRRRR (SEQ ID NO: 109) orRRRLHRIHRRQHRSCRRRKRR (SEQ ID NO: Ho). Other compounds suitable forforming a complex with the nucleic acid molecule of either formula (I),(Ia), (II), (IIa), (IIb), (IIIc) and/or (IIIb) according to theinvention as defined above according to the invention may be selectedfrom the adjuvant compounds as defined herein, without being limitedthereto.

In this context, “forming a complex” shall mean that the nucleic acidmolecule of either formula (I), (Ia), (II), (IIa), (IIb), (IIIc) and/or(IIIb) according to the invention as defined above is bound to astabilizing compound as defined above, e.g. a cationic polymer, cationicpeptides or polypeptides, etc. by forming a non-covalent complex betweennucleic acid and stabilizing compound. Herein, “non-covalent” means thata reversible association of nucleic acid and stabilizing compound isformed by non-covalent interactions of these molecules, wherein themolecules are associated together by some type of interaction ofelectrons, other than a covalent bond, e.g. by van der Waals-bonds, i.e.a weak electrostatic attraction arising from a nonspecific attractiveforce of both molecules. Association of the nucleic acid molecule ofeither formula (I), (Ia), (II), (IIa), (IIb), (IIIc) and/or (IIIb)according to the invention as defined above and the stabilizing compoundis in equilibrium with dissociation of that complex. Without being boundto any theory, it is expected that the equilibrium is intracellularlyshifted towards dissociated nucleic acid molecule of either formula (I),(Ia), (II), (IIa), (IIb), (IIIc) and/or (IIIb) according to theinvention as defined above and the stabilizing compound.

According to an embodiment, the nucleic acid molecule of either formula(I), (Ia), (II), (IIa), (IIb), (IIIc) and/or (IIIb) according to theinvention as defined above can be an immunostimulating agent, ifadministered without any other pharmaceutically active component, or maybe used as an adjuvant, if administered together with a pharmaceuticallyactive component, e.g. as a composition containing both thepharmaceutically active component and the adjuvant component (e.g. avaccine composition containing a specific antigen and a nucleic acidmolecule according to either formula (I), (Ia), (II), (IIa), (IIb),(IIIc) and/or (IIIb) according to the invention as defined above as anadjuvant).

A nucleic acid molecule of either formula (I), (Ia), (II), (IIa), (IIb),(IIIc) and/or (IIIb) according to the invention as defined above as an“immunostimulating agent” is preferably capable of triggering anon-antigen-specific, immune reaction (as provided by the innate immunesystem), preferably in an immunostimulating manner. An immune reactioncan generally be brought about in various ways. An important factor fora suitable immune response is the stimulation of different T-cellsub-populations. T-lymphocytes typically differentiate into twosub-populations, the T-helper 1 (Th1) cells and the T-helper 2 (Th2)cells, with which the immune system is capable of destroyingintracellular (Th1) and extracellular (Th2) pathogens (e.g. antigens).The two Th cell populations differ in the pattern of effector proteins(cytokines) produced by them. Thus, Th1 cells assist the cellular immuneresponse by activation of macrophages and cytotoxic T-cells. Th2 cells,on the other hand, promote the humoral immune response by stimulation ofB-cells for conversion into plasma cells and by formation of antibodies(e.g. against antigens). The Th1/Th2 ratio is therefore of greatimportance in the immune response. In connection with the presentinvention, the Th1/Th2 ratio of the immune response is preferablydisplaced by the immunostimulating agent, namely the nucleic acidmolecule of either formula (I), (Ia), (II), (IIa), (IIb), (IIIc) and/or(IIIb) according to the invention as defined above in the directiontowards the cellular response, that is to say the Th1 response, and apredominantly cellular immune response is thereby induced. As definedabove, the nucleic acid of the invention exerts by itself an unspecificimmune response, which allows the nucleic acid to be used as such(without adding another pharmaceutically active component) as animmunostimulating agent. If administered together with anotherpharmaceutically active component, preferably a specificallyimmunostimulating component, the nucleic acid of the invention serves asan adjuvant supporting the specific immune response elicited by theother pharmaceutically active component.

The present invention also relates to pharmaceutical compositionscontaining at least one inventive nucleic acid molecule of eitherformula (I), (Ia), (II), (IIa), (IIb), (IIIc) and/or (IIIb) according tothe invention as defined above and optionally a (compatible)pharmaceutically acceptable carrier and/or further auxiliary substancesand additives and/or adjuvants (first embodiment of an inventivecomposition). Moreover, the present invention relates to pharmaceuticalcompositions containing at least one nucleic acid molecule of eitherformula (I), (Ia), (II), (IIa), (IIb), (IIIc) and/or (IIIb) according tothe invention as defined above, e.g. one, two three, four six seven, ormore nucleic acid molecules thereof, a pharmaceutically active componentand optionally a pharmaceutically acceptable carrier and/or furtherauxiliary substances and additives and/or adjuvants (second embodimentof an inventive composition).

The pharmaceutical compositions according to the present inventiontypically comprise a safe and effective amount of at least one nucleicacid molecule of either formula (I), (Ia), (II), (IIa), (IIb), (IIIc)and/or (IIIb) according to the invention as defined above, or one, twothree, four six seven, or more nucleic acids thereof. As used here,“safe and effective amount” means an amount of each or all nucleic acidsof either formula (I), (Ia), (II), (IIa), (IIb), (IIIc) and/or (IIIb)according to the invention as defined above in the composition, that issufficient to significantly induce a positive modification of acondition to be treated, for example of a tumour, autoimmune diseases,allergies or infectious disease, etc. At the same time, however, a “safeand effective amount” is small enough to avoid serious side-effects,that are to say to permit a sensible relationship between advantage andrisk. The determination of these limits typically lies within the scopeof sensible medical judgment. In relation to the nucleic acid moleculeof either formula (I), (Ia), (II), (IIa), (IIb), (IIIc) and/or (IIIb)according to the invention as defined above, the expression “safe andeffective amount” preferably means an amount that is suitable forstimulating the immune system in such a manner that no excessive ordamaging immune reactions are achieved but, preferably, also no suchimmune reactions below a measurable level. A “safe and effective amount”of the nucleic acid molecule of either formula (I), (Ia), (II), (IIa),(IIb), (IIIc) and/or (IIIb) according to the invention as defined above,will vary in connection with the particular condition to be treated andalso with the age and physical condition of the patient to be treated,the severity of the condition, the duration of the treatment, the natureof the accompanying therapy, of the particular pharmaceuticallyacceptable carrier used, and similar factors, within the knowledge andexperience of the accompanying doctor. The pharmaceutical compositionsaccording to the invention can be used according to the invention forhuman and also for veterinary medical purposes.

According to the first embodiment, the above-described nucleic acidmolecule of either formula (I), (Ia), (II), (IIa), (IIb), (IIIc) and/or(IIIb) according to the invention as defined above, can by itself be theimmunostimulating agent (without addition of any other pharmaceuticallyactive components). This holds in particular, if the nucleic acidmolecule of either formula (I), (Ia), (II), (IIa), (IIb), (IIIc) and/or(IIIb) according to the invention as defined above contains a lipidmodification. The lipid may further enhance the immunostimulatoryproperties of the inventive nucleic acids or may well form atherapeutically active molecule, such as, for example, a vitamin, orsteroid, as described above, for example alpha-tocopherol (vitamin E),D-alpha-tocopherol, L-alpha-tocopherol, D,L-alpha-tocopherol, vitamin Esuccinate (VES), vitamin A and its derivatives, vitamin D and itsderivatives, vitamin K and its derivatives, etc.

The pharmaceutical composition according to the second embodiment of theinvention may contain (in addition to the at least one nucleic acidmolecule of either formula (I), (Ia), (II), (IIa), (IIb), (IIIc) and/or(IIIb) according to the invention as defined above) at least oneadditional pharmaceutically active component. A pharmaceutically activecomponent in this connection is a compound that has a therapeutic effectagainst a particular indication, preferably cancer diseases, autoimmunedisease, allergies or infectious diseases. Such compounds include,without implying any limitation, peptides, proteins, nucleic acids,(therapeutically active) low molecular weight organic or inorganiccompounds (molecular weight less than 5000, preferably less than 1000),sugars, antigens or antibodies, therapeutic agents already known in theprior art, antigenic cells, antigenic cellular fragments, cellularfractions; modified, attenuated or de-activated (e.g. chemically or byirridation) pathogens (virus, bacteria etc.) etc.

According to a first alternative of the second embodiment (of acomposition according to the invention), the pharmaceutically activecomponent contained in the pharmaceutical composition is animmunomodulatory component, preferably an immuno-stimulatory component.Most preferably, the pharmaceutically active component is an antigen orimmunogen. An “antigen” and an “immunogen” are to be understood as beingany structure that is able to bring about the formation of antibodiesand/or the activation of a cellular immune response, that is to say aspecific (and not an adjuvant) immune response. According to theinvention, therefore, the terms “antigen” and “immunogen” are usedsynonymously. Examples of antigens are peptides, polypeptides, that isto say also proteins, cells, cell extracts, polysaccharides,polysaccharide conjugates, lipids, glycolipids and carbohydrates. Therecome into consideration as antigens, for example, tumour antigens,animal, herbal, viral, bacterial, fungal and protozoological antigens,autoimmune antigens or allergens. Preference is given to surfaceantigens of tumour cells and surface antigens, in particular secretedforms, of viral, bacterial, fungal or protozoological pathogens. Theantigen can, of course, be present, for example in a vaccine accordingto the invention, also as a haptene coupled to a suitable carrier. Otherantigenic components, e.g. deactivated or attenuated pathogens (asdescribed above), may be used as well.

Antigenic (poly)peptides include all known antigenic peptides, forexample tumour antigens, etc. Specific examples of tumour antigens areinter alia tumour-specific surface antigens (TSSAs), for example 5T4,707-AP, 9D7, AFP, AlbZIP HPG1, alpha-5-beta-1-integrin,alpha-5-beta-6-integrin, alpha-actinin-4/m, alpha-methylacyl-coenzyme Aracemase, ART-4, ARTC1/m, B7H4, BAGE-1, BCL-2, bcr/abl, beta-catenin/m,BING-4, BRCA1/m, BRCA2/m, CA 15-3/CA 27-29, CA 19-9, CA72-4, CA125,calreticulin, CAMEL, CASP-8/m, cathepsin B, cathepsin L, CD19, CD20,CD22, CD25, CDE30, CD33, CD4, CD52, CD55, CD56, CD80, CDC27/m, CDK4/m,CDKN2A/m, CEA, CLCA2, CML28, CML66, COA-1/m, coactosin-like protein,collage XXIII, COX-2, CT-9/BRD6, Cten, cyclin B1, cyclin D1, cyp-B,CYPB1, DAM-10, DAM-6, DEK-CAN, EFTUD2/m, EGFR, ELF2/m, EMMPRIN, EpCam,EphA2, EphA3, ErbB3, ETV6-AML1, EZH2, FGF-5, FN, Frau-1, G250, GAGE-1,GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE7b, GAGE-8, GDEP, GnT-V,gp100, GPC3, GPNMB/m, HAGE, HAST-2, hepsin, Her2/neu, HERV-K-MEL,HLA-A*0201-R17I, HLA-A11/m, HLA-A2/m, HNE, homeobox NKX3.1,HOM-TES-14/SCP-1, HOM-TES-85, HPV-E6, HPV-E7, HSP70-2M, HST-2, hTERT,iCE, IGF-1R, IL-13Ra2, IL-2R, IL-5, immature laminin receptor,kallikrein-2, kallikrein-4, Ki67, KIAA0205, KIAA0205/m, KK-LC-1,K-Ras/m, LAGE-A1, LDLR-FUT, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6,MAGE-A9, MAGE-A10, MAGE-A12, MAGE-B1, MAGE-B2, MAGE-B3, MAGE-B4,MAGE-B5, MAGE-B6, MAGE-B10, MAGE-B16, MAGE-B17, MAGE-C1, MAGE-C2,MAGE-C3, MAGE-D1, MAGE-D2, MAGE-D4, MAGE-E1, MAGE-E2, MAGE-F1, MAGE-H1,MAGEL2, mammaglobin A, MART-1/melan-A, MART-2, MART-2/m, matrix protein22, MC1R, M-CSF, ME1/m, mesothelin, MG50/PXDN, MMP11, MN/CA IX-antigen,MRP-3, MUC-1, MUC-2, MUM-1/m, MUM-2/m, MUM-3/m, myosin class I/m,NA88-A, N-acetylglucosaminyltransferase-V, Neo-PAP, Neo-PAP/m, NFYC/m,NGEP, NMP22, NPM/ALK, N-Ras/m, NSE, NY-ESO-1, NY-ESO-B, OA1, OFA-iLRP,OGT, OGT/m, OS-9, OS-9/m, osteocalcin, osteopontin, p15, p190 minorbcr-abl, p53, p53/m, PAGE-4, PAI-1, PAI-2, PART-1, PATE, PDEF,Pim-1-Kinase, Pin-1, Pml/PARalpha, POTE, PRAME, PRDX5/m, prostein,proteinase-3, PSA, PSCA, PSGR, PSM, PSMA, PTPRK/m, RAGE-1, RBAF600/m,RHAMM/CD168, RU1, RU2, S-100, SAGE, SART-1, SART-2, SART-3, SCC,SIRT2/m, Sp17, SSX-1, SSX-2/HOM-MEL-40, SSX-4, STAMP-1, STEAP, survivin,survivin-2B, SYT-SSX-1, SYT-SSX-2, TA-90, TAG-72, TARP, TEL-AML1,TGFbeta, TGFbetaRII, TGM-4, TPI/m, TRAG-3, TRG, TRP-1, TRP-2/6b,TRP/INT2, TRP-p8, tyrosinase, UPA, VEGF, VEGFR-2/FLK-1, and WT1. Anyclass of tumor antigens is suitable for the purpose of the presentinvention, e.g. tumor antigens known to be involved inneovascularization, influencing the extracellular matrix structure etc.The tumor antigens may be provided in the pharmaceutical composition asprotein or peptide antigen or as mRNA or DNA encoding the tumor antigensor epitopes thereof, preferably the above tumor antigens.

By a second alternative of the second embodiment (for a compositionaccording to the invention containing the inventive nucleic acid (as anadjuvant) and the additional pharmaceutically active component) thepharmaceutically active component is an antibody. In this connection,any therapeutically suitable antibody can be used. Particular preferenceis given according to the invention to an antibody directed againstantigens, proteins or nucleic acids that play an important part incancer diseases or infectious diseases, for example cell surfaceproteins, tumour suppressor genes or inhibitors thereof, growth andelongation factors, apoptosis-relevant proteins, tumour antigens, orantigens as described hereinbefore, etc.

According to a third alternative of the second embodiment, thepharmaceutically active component contained in the pharmaceuticalcomposition according to the invention is a nucleic acid. Such a nucleicacid can be single-stranded or double-stranded and can be in the form ofa homo- or hetero-duplex and also in linear or circular form. A nucleicacid contained as a pharmaceutically active component in thepharmaceutical composition is not limited in terms of its length and caninclude any naturally occurring nucleic acid sequence or its complementor a fragment thereof. Likewise, the nucleic acid used in thisconnection can be partially or wholly of synthetic nature. For example,the nucleic acid can include a nucleic acid that codes for a(therapeutically relevant) protein and/or that is capable of bringingabout an immune reaction, for example an antigen or a nucleic acidcoding for an antigen. An antigen here is preferably an antigen asdescribed hereinbefore.

Preferably, the nucleic acid contained as a pharmaceutically activecomponent in the pharmaceutical composition according to the inventionis an mRNA. Such an mRNA can be added in its naked form to thepharmaceutical composition according to the invention or in a stabilizedform that reduces or even prevents the degradation of the nucleic acidin vivo, for example by exo- and/or endo-nucleases.

For example, the mRNA contained as a pharmaceutically active componentin the pharmaceutical composition according to the invention can bestabilized by an above-defined 5′ Cap, and alternatively or additionallyby a poly-A tail and/or a poly-C tail at the 3′ end of at least 50nucleotides, preferably at least 70 nucleotides, more preferably atleast 100 nucleotides, particularly preferably at least 200 nucleotides.As already mentioned, the terminal structure is of critical importancein vivo. The RNA is recognised as mRNA via these structures and thedegradation is regulated. In addition, however, there are furtherprocesses that stabilize or destabilize RNA. Many of these processes arestill unknown, but an interaction between the RNA and proteins oftenappears to be decisive therefor. For example, an “mRNA surveillancesystem” has recently been described (Hellerin and Parker, Ann. Rev.Genet. 1999, 33: 229 to 260), in which incomplete or non-sense mRNA isrecognised by particular feedback protein interactions in the cytosoland is made amenable to degradation, a majority of these processes beingcarried out by exonucleases.

The stabilization of the mRNA contained as a pharmaceutically activecomponent in the pharmaceutical composition according to the inventioncan likewise by carried out by associating or complexing the mRNA with,or binding it to, a cationic compound, in particular a polycationiccompound, for example a (poly)cationic peptide or protein. In particularthe use of protamine, nucleoline, spermin or spermidine as thepolycationic (nucleic-acid-binding) protein is particularly effective.Furthermore, the use of other cationic peptides or proteins, such aspoly-L-lysine or histones, is likewise possible. This procedure forstabilizing mRNA is described in EP-A-1083232, the disclosure of whichis incorporated by reference into the present invention in its entirety.Further preferred cationic substances which can be used for stabilizingthe mRNA present as a pharmaceutically active component include cationiccompounds as disclosed herein in connection with adjuvants, which aresuitable for depot and delivery of the inventive nucleic acid, e.g.cationic polysaccharides, for example chitosan, polybrene,polyethyleneimine (PEI) or poly-L-lysine (PLL), etc. Apart from theaction of the lipid-modified nucleic acid molecule of either formula(I), (Ia), (II), (IIa), (IIb), (IIIc) and/or (IIIb) in the form of anadjuvant in improving cell permeability, which is already advantageous,the association or complexing of the mRNA with cationic compounds, e.g.cationic proteins or cationic lipids, e.g. oligofectamine as a lipidbased complexation reagent) preferably increases the transfer of themRNA present as a pharmaceutically active component into the cells to betreated or into the organism to be treated. It is also referred to thedisclosure herein with regard to the stabilizing effect for the nucleicacid molecule of the invention by complexation, which holds for thestabilization of mRNA as well.

Another approach to stabilize mRNA as a pharmaceutically activecomponent in the pharmaceutical composition according to the inventionis the targeted changing of the sequence of the mRNA by removing orchanging so-called destabilizing sequence elements (DSEs). Signalproteins are able to bind to these destabilizing sequence elements(DSEs), which occur in eukaryotic mRNA in particular, and regulate theenzymatic degradation of the mRNA in vivo. Therefore, in order furtherto stabilize an mRNA present as a pharmaceutically active component, oneor more changes are preferably made as compared with the correspondingregion of the wild-type mRNA, so that no destabilizing sequence elementsare present. Of course, it is likewise preferred according to theinvention to eliminate DSEs optionally present in the untranslatedregions (3′- and/or 5′-UTR) from the mRNA. Examples of the above DSEsare AU-rich sequences (“AURES”), which occur in 3′-UTR sections ofnumerous unstable mRNAs (Caput et al., Proc. Natl. Acad. Sci. USA 1986,83: 1670 to 1674). The mRNA used as a pharmaceutically active componentis therefore preferably modified as compared with the wild-type mRNA insuch a manner that it does not contain any such destabilizing sequences.This is also true of those sequence motifs that are recognised bypossible endonucleases, for example the sequence GAACAAG, which iscontained in the 3′-UTR segment of the gene coding for the transferrinreceptor (Binder et al., EMBO J. 1994, 13: 1969 to 1980). Such sequencemotifs are preferably also eliminated from the lipid-modified nucleicacid molecule according to the invention.

The mRNA as a pharmaceutically active component in the pharmaceuticalcomposition according to the invention can further be modified, forexample for an efficient translation that may be desired, in such amanner that effective binding of the ribosomes to the ribosomal bindingsite (Kozak sequence: GCCGCCACCAUGG (SEQ ID NO: 111), the AUG forms thestart codon) takes place. It has been noted in this connection that anincreased A/U content around this position permits more efficientribosome binding to the mRNA.

Furthermore, it is possible to introduce one or more so-called IRES(internal ribosome entry side(s)) (sequences) into the mRNA used as apharmaceutically active component. An IRES can thus function as the onlyribosomal binding site, but it can also serve to provide an mRNA thatcodes for a plurality of peptides or polypeptides which are to betranslated independently of one another by the ribosomes(“multicistronic mRNA”). Examples of IRES sequences which can be usedaccording to the invention are those from picorna viruses (e.g. FMDV),plague viruses (CFFV), polio viruses (PV), encephalo-myocarditis viruses(ECMV), foot-and-mouth viruses (FMDV), hepatitis C viruses (HCV),conventional swine fever viruses (CSFV), murine leukoma virus (MLV),simean immune deficiency virus (SIV) or cricket paralysis viruses(CrPV).

The mRNA optionally used as a pharmaceutically active component in thepharmaceutical composition according to the invention can likewisecontain in its 5′- and/or 3′-untranslated regions stabilizing sequencesthat are capable of increasing the half-life of the mRNA in the cytosol.These stabilizing sequences can exhibit 100% sequence homology withnaturally occurring sequences that occur in viruses, bacteria andeukaryotes, but they can also be partially or wholly of syntheticnature. As examples of stabilizing sequences which can be used in thepresent invention there may be mentioned the untranslated sequences(UTR) of the beta-globin gene, for example of Homo sapiens or Xenopuslaevis. Another example of a stabilizing sequence has the generalformula (C/U)CCAN_(x)CCC(U/A)Py_(x)UC(C/U)CC (SEQ ID NO: 112), which iscontained in the 3′-UTR of the very stable mRNA that codes for α-globin,alpha-(I)-collagen, 15-lipoxygenase or for tyrosine-hydroxylase (seeHolcik et al., Proc. Natl. Acad. Sci. USA 1997, 94: 2410 to 2414). Ofcourse, such stabilizing sequences can be used individually or incombination with one another as well as in combination with otherstabilizing sequences known to a person skilled in the art.

In order to further increase an eventually desired translation, the mRNAused as a pharmaceutically active component can exhibit the followingmodifications as compared with a corresponding wild-type mRNA, whichmodifications can be present either as alternatives or in combinationwith one another. On the one hand, the G/C content of the region of themodified mRNA coding for a peptide or polypeptide can be greater thanthe G/C content of the coding region of the wild-type mRNA coding forthe peptide or polypeptide, the amino acid sequence coded for beingunmodified compared with the wild type. This modification is based onthe fact that, for an efficient translation of an mRNA, the stability ofthe mRNA as such is critical. The composition and sequence of thevarious nucleotides plays a large part thereby. In particular, sequenceshaving an increased G(guanosine (guanine))/C(cytidine (cytosine))content are more stable than sequences having an increased A(adenosine(adenine))/U(uridine (uracil)) content. According to the invention,therefore, while retaining the translated amino acid sequence, thecodons are varied as compared with the wild-type mRNA in such a mannerthat they contain more G/C nucleotides. Because several codons code forthe same amino acid (degeneracy of the genetic code) the codons that areadvantageous for the stability can be determined (alternative codonusage). In dependence on the amino acid to be coded for by the mRNA,different possibilities for the modification of the mRNA sequence ascompared to the wild-type sequence are possible. In the case of aminoacids coded for by codons that contain solely G or C nucleotides, nomodification of the codon is necessary. Accordingly, the codons for Pro(CCC or CCG), Arg (CGC or CGG), Ala (GCC or GCG) and Gly (GGC or GGG) donot require any change because no A or U is present. In the followingcases, the codons that contain A and/or U nucleotides are changed by thesubstitution of different codons that code for the same amino acids butdo not contain A and/or U. Examples are: the codons for Pro can bechanged from CCU or CCA to CCC or CCG; the codons for Arg can be changedfrom CGU or CGA or AGA or AGG to CGC or CGG; the codons for Ala can bechanged from GCU or GCA to GCC or GCG; the codons for Gly can be changedfrom GGU or GGA to GGC or GGG. In other cases, although A and Unucleotides cannot be eliminated from the codons, it is possible toreduce the A and U content by the use of codons that contain fewer Aand/or U nucleotides. For example: the codons for Phe can be changedfrom UUU to UUC; the codons for Leu can be changed from UUA, CUU or CUAto CUC or CUG; the codons for Ser can be changed from UCU or UCA or AGUto UCC, UCG or AGC; the codon for Tyr can be changed from UAU to UAC;the stop codon UAA can be changed to UAG or UGA; the codon for Cys canbe changed from UGU to UGC; the codon His can be changed from CAU toCAC; the codon for Gln can be changed from CAA to CAG; the codons forIle can be changed from AUU or AUA to AUC; the codons for Thr can bechanged from ACU or ACA to ACC or ACG; the codon for Asn can be changedfrom AAU to AAC; the codon for Lys can be changed from AAA to AAG; thecodons for Val can be changed from GUU or GUA to GUC or GUG; the codonfor Asp can be changed from GAU to GAC; the codon for Glu can be changedfrom GAA to GAG. In the case of the codons for Met (AUG) and Trp (UGG),on the other hand, there is no possibility of sequence modification. Thesubstitutions listed above can, of course, be used individually but alsoin all possible combinations for increasing the G/C content of themodified mRNA as compared with the original sequence. Thus, for example,all codons for Thr occurring in the original (wild-type) sequence can bechanged to ACC (or ACG). Preferably, however, combinations of the abovesubstitution possibilities are used, for example: substitution of allcodons in the original sequence coding for Thr to ACC (or ACG) andsubstitution of all codons originally coding for Ser to UCC (or UCG orAGC); substitution of all codons in the original sequence coding for Ileto AUC and substitution of all codons originally coding for Lys to AAGand substitution of all codons originally coding for Tyr to UAC;substitution of all codons in the original sequence coding for Val toGUC (or GUG) and substitution of all codons originally coding for Glu toGAG and substitution of all codons originally coding for Ala to GCC (orGCG) and substitution of all codons originally coding for Arg to CGC (orCGG); substitution of all codons in the original sequence coding for Valto GUC (or GUG) and substitution of all codons originally coding for Gluto GAG and substitution of all codons originally coding for Ala to GCC(or GCG) and substitution of all codons originally coding for Gly to GGC(or GGG) and substitution of all codons originally coding for Asn toAAC; substitution of all codons in the original sequence coding for Valto GUC (or GUG) and substitution of all codons originally coding for Pheto UUC and substitution of all codons originally coding for Cys to UGCand substitution of all codons originally coding for Leu to CUG (or CUC)and substitution of all codons originally coding for Gln to CAG andsubstitution of all codons originally coding for Pro to CCC (or CCG);etc. Preferably, the G/C content of the region (or of each other furthersection optionally present) of the mRNA that codes for the peptide orpolypeptide is increased by at least 7% points, more preferably by atleast 15% points, particularly preferably by at least 20% points, ascompared with the G/C content of the coded region of the wild-type mRNAcoding for the corresponding peptide or polypeptide and is preferably atleast 50%, more preferably at least 70% and most preferably at least90%. It is particularly preferred in this connection to increase the G/Ccontent of the mRNA so modified in comparison with the wild-typesequence to the maximum possible degree.

A further preferred modification of an mRNA used as a pharmaceuticallyactive component in the pharmaceutical composition is based on thefinding that the translation efficiency is also determined by adifferent frequency in the occurrence of tRNAs in cells. If, therefore,so-called “rare” codons are present in an increased number in an RNAsequence, then the corresponding mRNA is translated markedly more poorlythan in the case where codons coding for relatively “frequent” tRNAs arepresent. According to the invention, therefore, the coding region in themRNA used as a pharmaceutically active component is modified as comparedwith the corresponding region of the wild-type mRNA in such a mannerthat at least one codon of the wild-type sequence that codes for arelatively rare tRNA in the cell is replaced by a codon that codes for arelatively frequent tRNA in the cell, which carries the same amino acidas the relatively rare tRNA. By means of this modification, the RNAsequences are so modified that codons are introduced for whichfrequently occurring tRNAs are available. Which tRNAs occur relativelyfrequently in the cell and which, by contrast, are relatively rare isknown to a person skilled in the art; see, for example, Akashi, Curr.Opin. Genet. Dev. 2001, 11(6): 660-666. By means of this modification itis possible according to the invention to replace all codons of thewild-type sequence that code for a relatively rare tRNA in the cell by acodon that codes for a relatively frequent tRNA in the cell, whichcarries the same amino acid as the relatively rare tRNA. It isparticularly preferred to combine the increased, in particular maximum,sequential G/C content in the mRNA as described above with the“frequent” codons, without changing the amino acid sequence of anantigenic peptide or polypeptide (one or more) coded for by the codingregion of the mRNA. Preferred antigens, which may be coded by the G/Cenriched/optimized mRNA, are listed above.

According to a fourth alternative of the second embodiment (for thecomposition of the present invention), the nucleic acid contained as apharmaceutically active component in the pharmaceutical compositionaccording to the invention is a dsRNA, preferably a siRNA. A dsRNA, or asiRNA, is of interest particularly in connection with the phenomenon ofRNA interference. Attention was drawn to the phenomenon of RNAinterference in the course of immunological research. In recent years,an RNA-based defence mechanism has been discovered, which occurs both inthe kingdom of the fungi and in the plant and animal kingdom and acts asan “immune system of the genome”. The system was originally described invarious species independently of one another, first in C. elegans,before it was possible to identify the underlying mechanisms of theprocesses as being identical: RNA-mediated virus resistance in plants,PTGS (posttranscriptional gene silencing) in plants, and RNAinterference in eukaryotes are accordingly based on a common procedure.The in vitro technique of RNA interference (RNAi) is based ondouble-stranded RNA molecules (dsRNA), which trigger thesequence-specific suppression of gene expression (Zamore (2001) Nat.Struct. Biol. 9: 746-750; Sharp (2001) Genes Dev. 5:485-490: Hannon(2002) Nature 41: 244-251). In the transfection of mammalian cells withlong dsRNA, the activation of protein kinase R and RnaseL brings aboutunspecific effects, such as, for example, an interferon response (Starket al. (1998) Annu. Rev. Biochem. 67: 227-264; He and Katze (2002) ViralImmunol. 15: 95-119). These unspecific effects are avoided when shorter,for example 21- to 23-mer, so-called siRNA (small interfering RNA), isused, because unspecific effects are not triggered by siRNA that isshorter than 30 by (Elbashir et al. (2001) Nature 411: 494-498).Recently, dsRNA molecules have also been used in vivo (McCaffrey et al.(2002), Nature 418: 38-39; Xia et al. (2002), Nature Biotech. 20:1006-1010; Brummelkamp et al. (2002), Cancer Cell 2: 243-247).

The double-stranded RNA (dsRNA) eventually used as a pharmaceuticallyactive component in the pharmaceutical composition according to theinvention therefore preferably contains a sequence having the generalstructure 5′-(N₁₇₋₂₉)-3′, wherein N is any base and representsnucleotides. The general structure is composed of a double-stranded RNAhaving a macromolecule composed of ribonucleotides, the ribonucleotidecomprising a pentose (ribose or deoxyribose), an organic base and aphosphate. The organic bases in the RNA here comprise the purine basesadenosine (adenine) (A) and guanosine (guanine) (G) and of thepyrimidine bases cytidine (cytosine) (C) and uridine (uracil) (U). ThedsRNA eventually used as a pharmaceutically active component in thepharmaceutical composition according to the invention contains suchnucleotides or nucleotide analogues having an oriented structure. dsRNAsused as a pharmaceutically active component according to the inventionpreferably have the general structure 5′-(N₁₉₋₂₅)-3′, more preferably5′-(N₁₉₋₂₄)-3′, yet more preferably 5′-(N₂₁₋₂₃)-3′, wherein N is anybase. Preferably at least 90%, more preferably 95% and especially 100%of the nucleotides of a dsRNA used as a pharmaceutically activecomponent will be complementary to a section of the (m)RNA sequence of a(therapeutically relevant) protein or antigen described (as apharmaceutically active component) hereinbefore. 90% complementary meansthat with a length of a dsRNA used according to the invention of, forexample, 20 nucleotides, this contains not more than 2 nucleotideswithout corresponding complementarity with the corresponding section ofthe (m)RNA. The sequence of the double-stranded RNA optionally used inthe pharmaceutical composition according to the invention is, however,preferably wholly complementary in its general structure with a sectionof the (m)RNA of a protein or antigen described as a pharmaceuticallyactive component hereinbefore.

In principle, all the sections having a length of from 17 to 29,preferably from 19 to 25, base pairs that occur in the coding region ofthe (m)RNA can serve as target sequence for a dsRNA eventually used as apharmaceutically active component in the pharmaceutical compositionaccording to the invention. Equally, dsRNAs used as a pharmaceuticallyactive component can also be directed against nucleotide sequences of a(therapeutically relevant) protein or antigen described (as apharmaceutically active component) hereinbefore that do not lie in thecoding region, in particular in the 5′ non-coding region of the (m)RNA,for example, therefore, against non-coding regions of the (m)RNA havinga regulatory function. The target sequence of the dsRNA used as apharmaceutically active component of a protein or antigen describedhereinbefore can therefore lie in the translated and untranslated regionof the (m)RNA and/or in the region of the control elements. The targetsequence of a dsRNA used as a pharmaceutically active component in thepharmaceutical composition according to the invention can also lie inthe overlapping region of untranslated and translated sequence; inparticular, the target sequence can comprise at least one nucleotideupstream of the start triplet of the coding region of the (m)RNA.

A modified nucleotide can preferably occur in a dsRNA eventually used asa pharmaceutically active component in the pharmaceutical compositionaccording to the invention. The expression “modified nucleotide” meansaccording to the invention that the nucleotide in question has beenchemically modified. The person skilled in the art understands by theexpression “chemical modification” that the modified nucleotide has beenchanged in comparison with naturally occurring nucleotides by thereplacement, addition or removal of one or more atoms or atom groups. Atleast one modified nucleotide in dsRNA used according to the inventionserves on the one hand for stability and on the other hand to preventdissociation. Preferably, from 2 to 10 and more preferably from 2 to 5nucleotides in a dsRNA used according to the invention have beenmodified. Advantageously, at least one 2′-hydroxy group of thenucleotides of the dsRNA in the double-stranded structure has beenreplaced by a chemical group, preferably a 2′-amino or a 2′-methylgroup. At least one nucleotide in at least one strand of thedouble-stranded structure can also be a so-called “locked nucleotide”having a sugar ring that has been chemically modified, preferably by a2′-O, 4′-C-methylene bridge. Several nucleotides of the dsRNA usedaccording to the invention are advantageously locked nucleotides.Moreover, by modification of the backbone of a dsRNA used according tothe invention, premature degradation thereof can be prevented.Particular preference is given in this connection to a dsRNA that hasbeen modified in the form of phosphorothioate, 2′-O-methyl-RNA, LNA,LNA/DNA gapmers, etc. and therefore has a longer half-life in vivo.

The ends of the double-stranded RNA (dsRNA) used as a pharmaceuticallyactive component in the pharmaceutical composition according to theinvention can preferably be modified in order to counteract degradationin the cell or dissociation into the individual strands, in particularin order to avoid premature degradation by nucleases. A normallyundesirable dissociation of the individual strands of dsRNA occurs inparticular when low concentrations thereof or short chain lengths areused. For the particularly effective inhibition of dissociation, thecohesion, effected by the nucleotide pairs, of the double-strandedstructure of dsRNA used according to the invention can be increased byat least one, preferably more than one, chemical linkage(s). A dsRNAused as a pharmaceutically active component in the pharmaceuticalcomposition according to the invention whose dissociation has beenreduced has higher stability towards enzymatic and chemical degradationin the cell or in the organism (in vivo) or ex vivo and therefore has alonger half-life. A further possibility for preventing prematuredissociation in the cell of dsRNA used according to the inventionconsists in forming hairpin loop(s) at each end of the strands. In aparticular embodiment, a dsRNA used in the pharmaceutical compositionaccording to the invention therefore has a hairpin structure in order toslow the dissociation kinetics. In such a structure, a loop structure isformed preferably at the 5′ and/or 3′ end. Such a loop structure has nohydrogen bridges, and typically therefore no complementarity, betweennucleotide bases. Typically, such a loop has a length of at least 5,preferably at least 7 nucleotides and in that manner links the twocomplementary individual strands of a dsRNA used according to theinvention. In order to prevent dissociation of the strands, thenucleotides of the two strands of the dsRNA used according to theinvention can likewise preferably be so modified that strengthening ofthe hydrogen bridge bond is achieved, for example by increasing thehydrogen bridge bond capacity between the bases by optionally modifiednucleotides. As a result, the stability of the interaction between thestrands is increased and the dsRNA is protected against attack byRNases.

According to a particularly preferred embodiment, the dsRNA used as apharmaceutically active component in the pharmaceutical compositionaccording to the invention is directed against the (m)RNA of a proteinor antigen as described hereinbefore. The dsRNA used thereby preferablysuppresses the translation of an above-described protein or antigen in acell to the extent of at least 50%, more preferably 60%, yet morepreferably 70% and most preferably at least 90%, that is to say the cellcontains preferably not more than half of the naturally occurring(without treatment with dsRNA used according to the invention) cellularconcentration of an above-described protein or antigen. The suppressionof the translation of these proteins or antigens in cells after additionof dsRNA molecules used according to the invention is based on thephenomenon of RNA interference caused by such molecules. The dsRNA usedaccording to the invention is then a so-called siRNA, which triggers thephenomenon of RNA interference and can bind the (m)RNA of anabove-described protein or antigen. Measurement or demonstration of thetranslation suppression triggered in cells by the dsRNA used accordingto the invention can be carried out by Northern blot, quantitativereal-time PCR or, at protein level, with specific antibodies against anabove-described protein or antigen. The dsRNA eventually used as apharmaceutically active component in the pharmaceutical compositionaccording to the invention, and a corresponding siRNA, can be preparedby processes known to a person skilled in the art.

The pharmaceutical composition (according to the first or the secondembodiment) according to the invention typically contains a (compatible)pharmaceutically acceptable carrier. The expression “(compatible)pharmaceutically acceptable carrier” used here preferably includes theliquid or non-liquid basis of the composition. The term “compatible” asused herein means that the constituents of the pharmaceuticalcomposition are capable of being mixed with the pharmaceutically activecomponent, with the nucleic acid of the invention as immunostimulatingagent or as an adjuvant as such and with one another component in such amanner that no interaction occurs which would substantially reduce thepharmaceutical effectiveness of the composition under usual useconditions. Pharmaceutically acceptable carriers must, of course, havesufficiently high purity and sufficiently low toxicity to make themsuitable for administration to a person to be treated.

If the composition is provided in liquid form, the pharmaceuticallyacceptable carrier will typically comprise one or more (compatible)pharmaceutically acceptable liquid carriers. The composition maycomprise as (compatible) pharmaceutically acceptable liquid carrierse.g. pyrogen-free water; isotonic saline or buffered (aqueous)solutions, e.g phosphate, citrate etc. buffered solutions, vegetableoils, such as, for example, groundnut oil, cottonseed oil, sesame oil,olive oil, corn oil and oil from theobroma; polyols, such as, forexample, polypropylene glycol, glycerol, sorbitol, mannitol andpolyethylene glycol; alginic acid, etc. Particularly for injection ofthe inventive pharmaceutical composition, a buffer, preferably anaqueous buffer, may be used, containing a sodium salt, preferably atleast 50 mM of a sodium salt, a calcium salt, preferably at least 0.01mM of a calcium salt, and optionally a potassium salt, preferably atleast 3 mM of a potassium salt. According to a preferred embodiment, thesodium, calcium and, optionally, potassium salts may occur in the formof their halogenides, e.g. chlorides, iodides, or bromides, in the formof their hydroxides, carbonates, hydrogen carbonates, or sulfates, etc.Without being limited thereto, examples of sodium salts include e.g.NaCl, NaI, NaBr, Na₂CO₅, NaHCO₅, Na₂SO₄, examples of the optionalpotassium salts include e.g. KCl, KI, KBr, K₂CO₅, KHCO₅, K₂SO₄, andexamples of calcium salts include e.g. CaCl₂, CaI₂, CaBr₂, CaCO₅, CaSO₄,Ca(OH)₂. Furthermore, organic anions of the aforementioned cations maybe contained in the buffer. According to a more preferred embodiment,the buffer suitable for injection purposes as defined above, may containsalts selected from sodium chloride (NaCl), calcium chloride (CaCl₂) andoptionally potassium chloride (KCl), wherein further anions may bepresent additional to the chlorides. Typically, the salts in theinjection buffer are present in a concentration of at least 50 mM sodiumchloride (NaCl), at least 3 mM potassium chloride (KCl) and at least0.01 mM calcium chloride (CaCl₂). The injection buffer may behypertonic, isotonic or hypotonic with reference to the specificreference medium, i.e. the buffer may have a higher, identical or lowersalt content with reference to the specific reference medium, whereinpreferably such concentrations of the afore mentioned salts may be used,which do not lead to damage of cells due to osmosis or otherconcentration effects. Reference media are e.g. in “in vivo” methodsoccurring liquids such as blood, lymph, cytosolic liquids, or other bodyliquids, or e.g. liquids, which may be used as reference media in “invitro” methods, such as common buffers or liquids. Such common buffersor liquids are known to a skilled person. Ringer-Lactate solution isparticularly preferred as a liquid basis.

If the composition is provided in solid form, the pharmaceuticallyacceptable carrier will typically comprise one or more (compatible)pharmaceutically acceptable solid carriers. The composition may compriseas (compatible) pharmaceutically acceptable solid carriers e.g. one ormore compatible solid or liquid fillers or diluents or encapsulatingcompounds may be used as well, which are suitable for administration toa person. Some examples of such (compatible) pharmaceutically acceptablesolid carriers are e.g. sugars, such as, for example, lactose, glucoseand sucrose; starches, such as, for example, corn starch or potatostarch; cellulose and its derivatives, such as, for example, sodiumcarboxymethylcellulose, ethylcellulose, cellulose acetate; powderedtragacanth; malt; gelatin; tallow; solid glidants, such as, for example,stearic acid, magnesium stearate; calcium sulphate, etc.

The choice of a (compatible) pharmaceutically acceptable carrier isdetermined in principle by the manner in which the pharmaceuticalcomposition according to the invention is administered. Thepharmaceutical composition according to the invention can beadministered, for example, systemically. Routes for administrationinclude, for example, oral, subcutaneous, intravenous, intramuscular,intra-articular, intra-synovial, intrasternal, intrathecal,intrahepatic, intralesional, intracranial, transdermal, intradermal,intrapulmonal, intraperitoneal, intracardial, intraarterial, andsublingual topical and/or intranasal routes. The suitable amount of thepharmaceutical composition to be used can be determined by routineexperiments with animal models. Such models include, without implyingany limitation, rabbit, sheep, mouse, rat, dog and non-human primatemodels. Preferred unit dose forms for injection include sterilesolutions of water, physiological saline or mixtures thereof. The pH ofsuch solutions should be adjusted to about 7.4. Suitable carriers forinjection include hydrogels, devices for controlled or delayed release,polylactic acid and collagen matrices. Suitable pharmaceuticallyacceptable carriers for topical application include those, which aresuitable for use in lotions, creams, gels and the like. If the compoundis to be administered perorally, tablets, capsules and the like are thepreferred unit dose form. The pharmaceutically acceptable carriers forthe preparation of unit dose forms, which can be used for oraladministration are well known in the prior art. The choice thereof willdepend on secondary considerations such as taste, costs and storability,which are not critical for the purposes of the present invention, andcan be made without difficulty by a person skilled in the art.

In order to further increase the immunogenicity, the pharmaceuticalcomposition according to the invention can additionally contain one ormore auxiliary substances. A synergistic action of the nucleic acidmolecule of either formula (I), (Ia), (II), (IIa), (IIb), (IIIc) and/or(IIIb) according to the invention as defined above and of an auxiliarysubstance optionally additionally contained in the pharmaceuticalcomposition (and, eventually, a pharmaceutically active component) asdescribed above is preferably achieved thereby. Depending on the varioustypes of auxiliary substances, various mechanisms can come intoconsideration in this respect. For example, compounds that permit thematuration of dendritic cells (DCs), for example lipopolysaccharides,TNF-alpha or CD40 ligand, form a first class of suitable auxiliarysubstances. In general, it is possible to use as auxiliary substance anyagent that influences the immune system in the manner of a “dangersignal” (LPS, GP96, etc.) or cytokines, such as GM-CFS, which allow animmune response produced by the immunostimulating adjuvant according tothe invention to be enhanced and/or influenced in a targeted manner.Particularly preferred auxiliary substances are cytokines, such asmonokines, lymphokines, interleukins or chemokines, that promote theimmune response, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,IL-9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19,IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29,IL-30, IL-31, IL-32, IL-33, INF-alpha, IFN-beta, INF-gamma, GM-CSF,G-CSF, M-CSF, LT-beta or TNF-alpha, growth factors, such as hGH.

Further additives which may be included in the pharmaceutical)compositions according to the invention are emulsifiers, such as, forexample, Tween®; wetting agents, such as, for example, sodium laurylsulfate; colouring agents; taste-imparting agents, pharmaceuticalcarriers; tablet-forming agents; stabilizers; antioxidants;preservatives.

The pharmaceutical composition according to the invention (first(without a pharmaceutically active component) and second (with apharmaceutically active component) embodiment) can also additionallycontain an adjuvant. Accordingly, the nucleic acid molecule of eitherformula (I), (Ia), (II), (IIa), (IIb), (IIIc) and/or (IIIb) according tothe invention as defined above as an immunostimulating agent or as anadjuvant (for the second embodiment of the inventive pharmaceuticalcomposition), can be combined with further immunostimulatingagents/adjuvants. Within the scope of the present invention, suitableagents/adjuvants for these purposes are in particular those compoundsthat enhance (by one or more mechanisms) the biologicalproperty/properties of the (modified or unmodified) nucleic acidmolecule of either formula (I), (Ia), (II), (IIa), (IIb), (IIIc) and/or(IIIb) according to the invention, that is to say in particularsubstances that potentiate the immunostimulating action of the nucleicacid molecule of either formula (I), (Ia), (II), (IIa), (IIb), (IIIc)and/or (IIIb) according to the invention. Examples of agents/adjuvantswhich can be used according to the invention include, without implyingany limitation, stabilizing cationic peptides or polypeptides asdescribed above, such as protamine, nucleoline, spermine or spermidine,and cationic polysaccharides, in particular chitosan, TDM, MDP, muramyldipeptide, pluronics, alum solution, aluminium hydroxide, ADJUMER™(polyphosphazene); aluminium phosphate gel; glucans from algae;algammulin; aluminium hydroxide gel (alum); highly protein-adsorbingaluminium hydroxide gel; low viscosity aluminium hydroxide gel; AF orSPT (emulsion of squalane (5%), Tween 80 (0.2%), Pluronic L121 (1.25%),phosphate-buffered saline, pH 7.4); AVRIDINE™ (propanediamine); BAYR005™((N-(2-deoxy-2-L-leucylamino-b-D-glucopyranosyl)-N-octadecyldodecanoyl-amidehydroacetate); CALCITRIOL™ (1□,25-dihydroxy-vitamin D3); calciumphosphate gel; CAP™ (calcium phosphate nanoparticles); choleraholotoxin, cholera-toxin-A1-protein-A-D-fragment fusion protein,sub-unit B of the cholera toxin; CRL 1005 (block copolymer P1205);cytokine-containing liposomes; DDA (dimethyldioctadecylammoniumbromide); DHEA (dehydroepiandrosterone); DMPC(dimyristoylphosphatidylcholine); DMPG(dimyristoylphosphatidylglycerol); DOC/alum complex (deoxycholic acidsodium salt); Freund's complete adjuvant; Freund's incomplete adjuvant;gamma inulin; Gerbu adjuvant (mixture of i)N-acetylglucosaminyl-(P1-4)-N-acetylmuramyl-L-alanyl-D-glutamine (GMDP),ii) dimethyldioctadecylammonium chloride (DDA), iii) zinc-L-proline saltcomplex (ZnPro-8); GM-CSF); GMDP(N-acetylglucosaminyl-(b1-4)-N-acetylmuramyl-L-alanyl-D-isoglutamine);imiquimod (1-(2-methypropyl)-1H-imidazo[4,5-c]quinoline-4-amine);ImmTher™(N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L-Ala-glyceroldipalmitate); DRVs (immunoliposomes prepared fromdehydration-rehydration vesicles); interferon-gamma; interleukin-1beta;interleukin-2; interleukin-7; interleukin-12; ISCOMS™(“Immunostimulating Complexes”); ISCOPREP 7.0.3.™; liposomes;LOXORIBINE™ (7-allyl-8-oxoguanosine (guanine)); LT oral adjuvant (E.coli labile enterotoxin-protoxin); microspheres and microparticles ofany composition; MF59™; (squalene-water emulsion); MONTANIDE ISA 51™(purified incomplete Freund's adjuvant); MONTANIDE ISA 720™(metabolisable oil adjuvant); MPL™ (3-Q-desacyl-4′-monophosphoryl lipidA); MTP-PE and MTP-PE liposomes((N-acetyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1,2-dipalmitoyl-sn-glycero-3-(hydroxyphosphoryloxy))ethylamide,monosodium salt); MURAMETIDE™ (Nac-Mur-L-Ala-D-Gln-OCH₃); MURAPALMITINE™and D-MURAPALMITINE™ (Nac-Mur-L-Thr-D-isoGln-sn-glyceroldipalmitoyl);NAGO (neuraminidase-galactose oxidase); nanospheres or nanoparticles ofany composition; NISVs (non-ionic surfactant vesicles); PLEURAN™(beta-glucan); PLGA, PGA and PLA (homo- and co-polymers of lactic acidand glycolic acid; micro-/nano-spheres); PLURONIC L121™; PMMA(polymethyl methacrylate); PODDS™ (proteinoid microspheres);polyethylene carbamate derivatives; poly-rA: poly-rU (polyadenylicacid-polyuridylic acid complex); polysorbate 80 (Tween 80); proteincochleates (Avanti Polar Lipids, Inc., Alabaster, Ala.); STIMULON™(QS-21); Quil-A (Quil-A saponin); S-28463(4-amino-otec-dimethyl-2-ethoxymethyl-1H-imidazo[4,5-c]quinoline-1-ethanol);SAF-1™ (“Syntex adjuvant formulation”); Sendai proteoliposomes andSendai-containing lipid matrices; Span-85 (sorbitan trioleate); Specol(emulsion of Marcol 52, Span 85 and Tween 85); squalene or Robane®(2,6,10,15,19,23-hexamethyltetracosan and2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexane);stearyltyrosine (octadecyltyrosine hydrochloride); Theramid®(N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L-Ala-dipalmitoxypropylamide);Theronyl-MDP (Termurtide™ or [thr 1]-MDP;N-acetylmuramyl-L-threonyl-D-isoglutamine); Ty particles (Ty-VLPs orvirus-like particles); Walter-Reed liposomes (liposomes containing lipidA adsorbed on aluminium hydroxide), and the like, etc. Lipopeptides,such as Pam3Cys, are likewise particularly suitable for combining withthe inventive nucleic acid molecule of either formula (I), (Ia), (II),(IIa), (IIb), (IIIc) and/or (IIIb) according to the invention as definedabove, in the form of an immunostimulating adjuvant (see Deres et al.,Nature 1989, 342: 561-564).

Adjuvants as mentioned above may be categorized into several classes,including adjuvants suitable for depot and delivery, for costimulation,adjuvants suitable as antagonists, etc. Preferred adjuvants suitable fordepot and delivery may include e.g. aluminium salts such as Adju-phos,Alhydrogel, Rehydragel, etc., emulsions, such as CFA, SAF, IFA, MF59,Provax, TiterMax, Montanide, Vaxfectin, etc., copolymers, such asOptivax (CRL1005), L121, Poloaxmer4010), etc., liposomes, such asStealth, etc., cochleates, such as BIORAL, etc., plant derivedadjuvatns, such as Q521, Quil A, Iscomatrix, ISCOM, etc. Preferredadjuvants suitable for costimulation may include e.g. Tomatine,biopolymers, such as PLG, PMM, Inulin, etc., Microbe derived adjuvants,such as Romurtide, DETOX, MPL, CWS, Mannose, CpG7909, ISS-1018, IC31,Imidazoquinolines, Ampligen, Ribi529, IMOxine, IRIVs, VLPs, choleratoxin, heat-labile toxin, Pam3Cys, Flagellin, GPI anchor, LNFPIII/LewisX, antimicrobial peptides, UC-1V150, RSV fusion protein, cdiGMP, etc.Preferred adjuvants suitable as antagonists may, e.g., include CGRPneuropeptide, etc.

Particularly preferred as adjuvants suitable for depot and delivery arecationic or polycationic compounds, including protamine, nucleoline,spermin or spermidine, or other cationic peptides or proteins, such aspoly-L-lysine (PLL), poly-arginine, basic polypeptides, cell penetratingpeptides (CPPs), including HIV-binding peptides, Tat, HIV-1 Tat (HIV),Tat-derived peptides, Penetratin, VP22 derived or analog peptides, HSVVP22 (Herpes simplex), MAP, KALA or protein transduction domains (PTDs,PpT620, prolin-rich peptides, arginine-rich peptides, lysine-richpeptides, MPG-peptide(s), Pep-1, L-oligomers, Calcitonin peptide(s),Antennapedia-derived peptides (particularly from Drosophilaantennapedia), pAntp, pIsl, FGF, Lactoferrin, Transportan, Buforin-2,Bac715-24, SynB, SynB(1), pVEC, hCT-derived peptides, SAP, protamine,spermine, spermidine, or histones. Additionally, preferred cationic orpolycationic proteins or peptides may be selected from followingproteins or peptides having the following total formula:(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x), whereinl+m+n+o+x=8-15, and l, m, n or o independently of each other may be anynumber selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or15, provided that the overall content of Arg, Lys, His and Ornrepresents at least 50% of all amino acids of the oligopeptide; and Xaamay be any amino acid selected from native (=naturally occurring) ornon-native amino acids except of Arg, Lys, His or Orn; and x may be anynumber selected from 0, 1, 2, 3 or 4, provided, that the overall contentof Xaa does not exceed 50% of all amino acids of the oligopeptide.Particularly preferred oligoarginines in this context are e.g. Arg₇,Arg₈, Arg₉, Arg₇, H₅R₉, R₉H₅, H₅R₉H₅, YSSR₉SSY, (RKH)₄, Y(RKH)₂R, etc.Further preferred cationic or polycationic compounds, which can be usedas adjuvant may include cationic polysaccharides, for example chitosan,polybrene, cationic polymers, e.g. polyethyleneimine (PEI), cationiclipids, e.g. DOTMA: [1-(2,3-sioleyloxy)propyl)]-N,N,N-trimethylammoniumchloride, DMRIE, di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP,DOPC, DODAP, DOPE: Dioleyl phosphatidylethanol-amine, DOSPA, DODAB,DOIC, DMEPC, DOGS: Dioctadecylamidoglicylspermin, DIMRI:Dimyristo-oxypropyl dimethyl hydroxyethyl ammonium bromide, DOTAP:dioleoyloxy-3-(trimethylammonio)propane, DC-6-14:O,O-ditetradecanoyl-N-(α-trimethylammonioacetyl)diethanolamine chloride,CLIP1: rac-[(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammoniumchloride, CLIP6:rac-[2(2,3-dihexadecyloxypropyl-oxymethyloxy)ethyl]trimethylammonium,CLIP9:rac-[2(2,3-dihexadecyloxypropyl-oxysuccinyloxy)ethyl]-trimethylammonium,oligofectamine, or cationic or polycationic polymers, e.g. modifiedpolyaminoacids, such as β-aminoacid-polymers or reversed polyamides,etc., modified polyethylenes, such as PVP(poly(N-ethyl-4-vinylpyridinium bromide)), etc., modified acrylates,such as pDMAEMA (poly(dimethylaminoethyl methylacrylate)), etc.,modified Amidoamines such as pAMAM (poly(amidoamine)), etc., modifiedpolybetaaminoester (PBAE), such as diamine end modified 1,4 butanedioldiacrylate-co-5-amino-1-pentanol polymers, etc., dendrimers, such aspolypropylamine dendrimers or pAMAM based dendrimers, etc.,polyimine(s), such as PEI: poly(ethyleneimine), poly(propyleneimine),etc., polyallylamine, sugar backbone based polymers, such ascyclodextrin based polymers, dextran based polymers, Chitosan, etc.,silan backbone based polymers, such as PMOXA-PDMS copolymers, etc.,Blockpolymers consisting of a combination of one or more cationic blocks(e.g. selected og a cationic polymer as mentioned above) and of one ormore hydrophilic- or hydrophobic blocks (e.g polyethyleneglycole); etc.Association or complexing the inventive nucleic acid molecule accordingto either formula (I), (Ia), (II), (IIa), (IIb), (IIIc) and/or (IIIb)according to the invention as defined above with cationic orpolycationic compounds preferably provides adjuvant properties to thenucleic acid and confers a stabilizing effect to the nucleic acid bycomplexation. The procedure for stabilizing the inventive nucleic acidis in general described in EP-A-1083232, the disclosure of which isincorporated by reference into the present invention in its entirety.Particularly preferred as cationic or polycationic compounds arecompounds selected from the group consisting of protamine, nucleoline,spermin, spermidine, oligoarginines as defined above, such as Arg₇,Arg₈, Arg₉, Arg₇, H₅R₉, R₉H₅, H₅R₉H₅, YSSR₉SSY, (RKH)₄, Y(RKH)₂R, etc.

Adjuvants which may have a costimulating effect include nucleic acids offormula (IV): G_(l)X_(m)G_(n), wherein: G is guanosine (guanine),uridine (uracil) or an analogue of guanosine (guanine) or uridine(uracil); X is guanosine (guanine), uridine (uracil), adenosine(adenine), thymidine (thymine), cytidine (cytosine) or an analogue ofthe above-mentioned nucleotides; 1 is an integer from 1 to 40, whereinwhen l=1 G is guanosine (guanine) or an analogue thereof, when l>1 atleast 50% of the nucleotides are guanosine (guanine) or an analoguethereof; m is an integer and is at least 3; wherein when m=3 X isuridine (uracil) or an analogue thereof, when m>3 at least 3 successiveuridines (uracils) or analogues of uridine (uracil) occur; n is aninteger from 1 to 40, wherein when n=1 G is guanosine (guanine) or ananalogue thereof, when n>1 at least 50% of the nucleotides are guanosine(guanine) or an analogue thereof;

or nucleic acids of formula (V): C_(l)X_(m)C_(n), wherein: C is cytidine(cytosine), uridine (uracil) or an analogue of cytidine (cytosine) oruridine (uracil); X is guanosine (guanine), uridine (uracil), adenosine(adenine), thymidine (thymine), cytidine (cytosine) or an analogue ofthe above-mentioned nucleotides; l is an integer from 1 to 40, whereinwhen l=1 C is cytidine (cytosine) or an analogue thereof, when l>1 atleast 50% of the nucleotides are cytidine (cytosine) or an analoguethereof; m is an integer and is at least 3; wherein when m=3 X isuridine (uracil) or an analogue thereof, when m>3 at least 3 successiveuridines (uracils) or analogues of uridine (uracil) occur; n is aninteger from 1 to 40, wherein when n=1 C is cytidine (cytosine) or ananalogue thereof, when n>1 at least 50% of the nucleotides are cytidine(cytosine) or an analogue thereof.

Any compound, which is known to be immunostimulating due to its bindingaffinity (as ligands) to Toll-like receptors: TLR1, TLR2, TLR3, TLR4,TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 or TLR13 may suitablybe used as further component to further stimulate the immune responseinduced by nucleic acids of the invention in the inventivepharmaceutical compositions.

Another class of compounds, which may be added to a pharmaceuticalcomposition of the invention, are CpG nucleic acids, in particularCpG-RNA or CpG-DNA. A CpG-RNA or CpG-DNA can be a single-strandedCpG-DNA (ss CpG-DNA), a double-stranded CpG-DNA (dsDNA), asingle-stranded CpG-RNA (ss CpG-RNA) or a double-stranded CpG-RNA (dsCpG-RNA). The CpG nucleic acid is preferably in the form of CpG-RNA,more preferably in the form of single-stranded CpG-RNA (ss CpG-RNA). TheCpG nucleic acid preferably contains at least one or more (mitogenic)cytidine (cytosine)/guanine dinucleotide sequence(s) (CpG motif(s)).According to a first preferred alternative, at least one CpG motifcontained in these sequences, that is to say the C (cytidine (cytosine))and the G (guanine) of the CpG motif, is unmethylated. All furthercytidines (cytosines) or guanines optionally contained in thesesequences can be either methylated or unmethylated. According to afurther preferred alternative, however, the C (cytidine (cytosine)) andthe G (guanine) of the CpG motif can also be present in methylated form.

According to a particularly preferred embodiment, the pharmaceuticalcomposition according to the invention can also be provided as avaccine. Vaccines according to the invention typically comprise(correspond to) a pharmaceutical composition according to the invention.The composition of such vaccines according to the invention ischaracterized by a specific class of pharmaceutically active componentsincorporated into the vaccine composition. Typically, thepharmaceutically active compound will be an immunostimulatory substance,which evokes a specific (adaptive) immune response against a certainantigen/s. The specific (adaptive) immune response elicited allows thesubject to develop an immune response (evoked by an active or passivemode) against e.g. a specific pathogen or a specific tumor.

The inventive pharmaceutical composition and, in particular theinventive vaccine, is specifically characterized by the manner in whichit is administered. Typically, pharmaceutical compositions of theinvention, in particular vaccines, are preferably administeredsystemically. Routes for the administration of such an inventivepharmaceutical composition/vaccine typically include oral, subcutaneous,intravenous, intramuscular, intra-articular, intra-synovial,intrasternal, intrathecal, intrahepatic, intralesional, intracranial,transdermal, intradermal, intrapulmonal, intraperitoneal, intracardial,intraarterial, and sublingual topical and/or intranasal routes.Alternatively, vaccines or pharmaceutical composition of the inventionmay be administered by an intradermal, subcutaneous, intramuscularroute. Compositions/vaccines are therefore formulated preferably inliquid or solid form as defined above for pharmaceutical compositions ingeneral. Further auxiliary substances (as defined above) can furtherincrease the immunogenicity, in particular of the vaccine, which maypreferably be incorporated into a vaccine according to the invention.Advantageously, one or more such auxiliary substances as definedhereinbefore is/are to be chosen, depending on the immunogenicity andother properties of the pharmaceutically active component in the vaccineaccording to the invention.

According to a further preferred object of the present invention, thepharmaceutical composition according to the invention, particularlypreferably the inventive vaccine, are used for the treatment ofindications mentioned by way of example hereinbelow. With apharmaceutical composition according to the invention, particularlypreferably an inventive vaccine, it is possible to treat, for example,diseases or conditions that are associated with various pathologicallyabsent immune responses or that require an immune response, preferablyan increased immune response, within the context of a therapy, forexample tumour-specific or pathogen-specific diseases, infectiousdiseases, etc or diseases, which may be treated by shifting the(exceeding) immune response to a TH1 dominated immune response and/or bydesensitizing the patient suffering from an exceeding immune response,as e.g. in allergies or autoimmune diseases. The production of such animmune response, or the increase of an already existing but optionallyinadequate immune response, by the pharmaceutical composition accordingto the invention is based substantially on its ability to trigger an anon-antigen-specific immune reaction. An important factor for a suitableimmune response is the stimulation of different T-cell sub-populations.T-lymphocytes typically differentiate into two sub-populations, theT-helper 1 (Th1) cells and the T-helper 2 (Th2) cells, with which theimmune system is capable of destroying intracellular (Th1) andextracellular (Th2) pathogens (e.g. antigens). The two Th cellpopulations differ in the pattern of the effector proteins (cytokines)produced by them. Thus, Th1 cells assist the cellular immune response byactivation of macrophages and cytotoxic T-cells. Th2 cells, on the otherhand, promote the humoral immune response by stimulation of the B-cellsfor conversion into plasma cells and by formation of antibodies (e.g.against antigens). The Th1/Th2 ratio is therefore of great importance inthe immune response. In connection with the present invention, theTh1/Th2 ratio of the immune response is preferably displaced by thepharmaceutical composition according to the invention containing atleast one nucleic acid molecule of either formula (I), (Ia), (II),(IIa), (IIb), (IIIc) and/or (IIIb) according to the invention as definedabove, e.g. one, two three, four six seven, or more nucleic acidsthereof, in the direction towards the cellular response, that is to saythe Th1 response, and a predominantly cellular immune response isthereby induced. Only by this displacement and the preferential, or evenexclusive, occurrence of a TH1 immune response an efficient treatment ofthe above-mentioned indications is possible. Preferably, therefore, thepresent pharmaceutical compositions or vaccines according to theinvention are used to trigger tumour-specific or pathogen-specificimmune responses. Such pharmaceutical compositions or vaccines accordingto the invention can be used particularly preferably for increasingimmune responses of antigen-presenting cells (APCs). Likewiseparticularly preferably, the pharmaceutical compositions or vaccinesaccording to the invention can be used for the treatment of cancer ortumour diseases, preferably selected from colon carcinomas, melanomas,renal carcinomas, lymphomas, acute myeloid leukaemia (AML), acutelymphoid leukaemia (ALL), chronic myeloid leukaemia (CML), chroniclymphocytic leukaemia (CLL), gastrointestinal tumours, pulmonarycarcinomas, gliomas, thyroid tumours, mammary carcinomas, prostatetumours, hepatomas, various virus-induced tumours such as, for example,papilloma virus-induced carcinomas (e.g. cervical carcinoma),adenocarcinomas, herpes virus-induced tumours (e.g. Burkitt's lymphoma,EBV-induced B-cell lymphoma), hepatitis B-induced tumours (hepatocellcarcinoma), HTLV-1- and HTLV-2-induced lymphomas, acousticneuromas/neurinomas, cervical cancer, lung cancer, pharyngeal cancer,anal carcinomas, glioblastomas, lymphomas, rectal carcinomas,astrocytomas, brain tumours, stomach cancer, retinoblastomas,basaliomas, brain metastases, medulloblastomas, vaginal cancer,pancreatic cancer, testicular cancer, melanomas, thyroidal carcinomas,bladder cancer, Hodgkin's syndrome, meningiomas, Schneeberger disease,bronchial carcinomas, hypophysis tumour, Mycosis fungoides, oesophagealcancer, breast cancer, carcinoids, neurinomas, spinaliomas, Burkitt'slymphomas, laryngeal cancer, renal cancer, thymomas, corpus carcinomas,bone cancer, non-Hodgkin's lymphomas, urethral cancer, CUP syndrome,head/neck tumours, oligodendrogliomas, vulval cancer, intestinal cancer,colon carcinomas, oesophageal carcinomas, wart involvement, tumours ofthe small intestine, craniopharyngeomas, ovarian carcinomas, soft tissuetumours/sarcomas, ovarian cancer, liver cancer, pancreatic carcinomas,cervical carcinomas, endometrial carcinomas, liver metastases, penilecancer, tongue cancer, gall bladder cancer, leukaemia, plasmocytomas,uterine cancer, lid tumour, prostate cancer, etc. It is particularlypreferred, if the lipid used in the lipid-modified nucleic acid or aspharmaceutically active component in the composition is alpha-tocopherol(vitamin E), D-alpha-tocopherol, L-alpha-tocopherol,D,L-alpha-tocopherol or vitamin E succinate (VES). alpha-Tocopherol(vitamin E) is not very toxic and exhibits potent anti-tumour activity(A. Bendich, L. J. Machlin Am. J. Clin. Nutr. 48 (1988) 612), whichmakes it appear very promising in cancer therapy. As an explanation forthe inhibition of the proliferation of tumour cells or the cytotoxicactivity thereon, two mechanisms inter alia are known: On the one hand,vitamin E is a potent antioxidant and a good radical acceptor (C. BorekAnn. NT Acad. Sci. 570 (1990) 417); on the other hand, it is able, bystimulating the immune response, to prevent tumour growth (G. Shklar, J.Schwartz, D. P. Trickler, S. Reid J. Oral Pathol. Med. 19 (1990) 60). Inmore recent works, a connection has further been found between theexpression of the tumour suppressor gene p53 in tumour cells (oralsquamous cancer) and treatment with vitamin E succinate (VES) (J.Schwartz, G. Shklar, D. Trickler Oral Oncol. Europ. J. Cancer 29B (1993)313). It has thereby been possible to observe both a stimulation of theproduction of wild-type p53, which acts as a tumour suppressor, and areduction in mutated p53, which develops oncogenic activity.Interestingly, the biological activity of VES on these tumour cells isdose-dependent in two respects: in physiological doses (0.001 to 50μmol/l), increasing cell growth is to be observed; in pharmacologicaldoses (100 to 154 μmol/l), cell growth is inhibited. This has been shownin cell culture (T. M. A. Elattar, A. S. Virji Anticancer Res. 19 (1999)365). It has also been possible to induce apoptosis in various breastcancer cell lines by treatment with VES (W. Yu, K. Israel, Q. Y. Liao,C. M. Aldaz, B. G. Sanders, K. Kline Cancer Res. 59 (1999) 953). Theinduced apoptosis is initiated via an interaction of Fas ligand and Fasreceptor. This is to be particularly emphasised because it has hithertonot been possible to observe such a mechanism in the corresponding celllines. There are various isomers of vitamin E, which differ in thenumber and position of the methyl groups on the aromatic ring. In thedescribed works, the biologically most active form of naturallyoccurring vitamin E, α-tocopherol, was used. This in turn occurs invarious stereoisomers, because the molecule contains three opticallyactive centres. The natural form of vitamin E is RRR-alpha-tocopherol(formerly D-alpha-tocopherol), but the racemate (D,L-alpha-tocopherol)is predominantly used nowadays. All the above-mentioned forms of vitaminE are likewise included as lipid within the scope of the presentinvention.

Likewise particularly preferably, at least one nucleic acid molecule ofeither formula (I), (Ia), (II), (IIa), (IIb), (IIIc) and/or (IIIb)according to the invention as defined above, or the pharmaceuticalcomposition according to the invention, are used for the treatment ofinfectious diseases. Without implying any limitation, such infectiousdiseases are preferably selected from influenza, malaria, SARS, yellowfever, AIDS, Lyme borreliosis, Leishmaniasis, anthrax, meningitis, viralinfectious diseases such as AIDS, Condyloma acuminata, hollow warts,Dengue fever, three-day fever, Ebola virus, cold, early summermeningoencephalitis (FSME), flu, shingles, hepatitis, herpes simplextype I, herpes simplex type II, Herpes zoster, influenza, Japaneseencephalitis, Lassa fever, Marburg virus, measles, foot-and-mouthdisease, mononucleosis, mumps, Norwalk virus infection, Pfeiffer'sglandular fever, smallpox, polio (childhood lameness), pseudo-croup,fifth disease, rabies, warts, West Nile fever, chickenpox, cytomegalicvirus (CMV), from bacterial infectious diseases such as miscarriage(prostate inflammation), anthrax, appendicitis, borreliosis, botulism,Camphylobacter, Chlamydia trachomatis (inflammation of the urethra,conjunctivitis), cholera, diphtheria, donavanosis, epiglottitis, typhusfever, gas gangrene, gonorrhoea, rabbit fever, Heliobacter pylori,whooping cough, climatic bubo, osteomyelitis, Legionnaire's disease,leprosy, listeriosis, pneumonia, meningitis, bacterial meningitis,anthrax, otitis media, Mycoplasma hominis, neonatal sepsis(Chorioamnionitis), noma, paratyphus, plague, Reiter's syndrome, RockyMountain spotted fever, Salmonella paratyphus, Salmonella typhus,scarlet fever, syphilis, tetanus, tripper, tsutsugamushi disease,tuberculosis, typhus, vaginitis (colpitis), soft chancre, and frominfectious diseases caused by parasites, protozoa or fungi, such asamoebiasis, bilharziosis, Chagas disease, athlete's foot, yeast fungusspots, scabies, malaria, onchocercosis (river blindness), or fungaldiseases, toxoplasmosis, trichomoniasis, trypanosomiasis (sleepingsickness), visceral Leishmaniosis, nappy/diaper dermatitis,schistosomiasis, fish poisoning (Ciguatera), candidosis, cutaneousLeishmaniosis, lambliasis (giardiasis), or sleeping sickness, or frominfectious diseases caused by Echinococcus, fish tapeworm, fox tapeworm,canine tapeworm, lice, bovine tapeworm, porcine tapeworm, miniaturetapeworm.

Accordingly, at least one nucleic acid of the invention of eitherformula (I), (Ia), (II), (IIa), (IIb), (IIIc) and/or (IIIb) according tothe invention as defined above, or the pharmaceutical composition of theinvention may be used for the preparation of a medicament for thetreatment of an allergic disorder or disease. Allergy is a conditionthat typically involves an abnormal, acquired immunologicalhypersensitivity to certain foreign antigens or allergens. Allergiesnormally result in a local or systemic inflammatory response to theseantigens or allergens and leading to immunity in the body against theseallergens. Allergens in this context include e.g. grasses, pollens,molds, drugs, or numerous environmental triggers, etc. Without beingbound to theory, several different disease mechanisms are supposed to beinvolved in the development of allergies. According to a classificationscheme by P. Gell and R. Coombs the word “allergy” was restricted totype I hypersensitivities, which are caused by the classical IgEmechanism. Type I hypersensitivity is characterised by excessiveactivation of mast cells and basophils by IgE, resulting in a systemicinflammatory response that can result in symptoms as benign as a runnynose, to life-threatening anaphylactic shock and death. Well known typesof allergies include, without being limited thereto, allergic asthma(leading to swelling of the nasal mucosa), allergic conjunctivitis(leading to redness and itching of the conjunctiva), allergic rhinitis(“hay fever”), anaphylaxis, angiodema, atopic dermatitis (eczema),urticaria (hives), eosinophilia, respiratory, allergies to insectstings, skin allergies (leading to and including various rashes, such aseczema, hives (urticaria) and (contact) dermatitis), food allergies,allergies to medicine, etc. With regard to the present invention, e.g.an inventive pharmaceutical composition or vaccine is provided, whichcontains an allergen (e.g. from a cat allergen, a dust allergen, a miteantigen, a plant antigen (e.g. a birch antigen) etc.) either as aprotein, an mRNA (or DNA) encoding for that protein allergen incombination with a nucleic acid of the invention of either formula (I),(Ia), (II), (IIa), (IIb), (IIIc) and/or (IIIb) according to theinvention as defined above. A pharmaceutical composition of the presentinvention may shift the (exceeding) immune response to a stronger TH1response, thereby suppressing or attenuating the undesired IgE response.

Likewise, at least one nucleic acid of the invention of either formula(I), (Ia), (II), (IIa), (IIb), (IIIc) and/or (IIIb) according to theinvention as defined above, or the pharmaceutically active compositionof the invention may be used for the preparation of a medicament for thetreatment of autoimmune diseases. Autoimmune diseases can be broadlydivided into systemic and organ-specific or localised autoimmunedisorders, depending on the principal clinico-pathologic features ofeach disease. Autoimmune disease may be divided into the categories ofsystemic syndromes, including SLE, Sjögren's syndrome, Scleroderma,Rheumatoid Arthritis and polymyositis or local syndromes which may beendocrinologic (DM Type 1, Hashimoto's thyroiditis, Addison's diseaseetc.), dermatologic (pemphigus vulgaris), haematologic (autoimmunehaemolytic anaemia), neural (multiple sclerosis) or can involvevirtually any circumscribed mass of body tissue. The autoimmune diseasesto be treated may be selected from the group consisting of type Iautoimmune diseases or type II autoimmune diseases or type IIIautoimmune diseases or type IV autoimmune diseases, such as, forexample, multiple sclerosis (MS), rheumatoid arthritis, diabetes, type Idiabetes (Diabetes mellitus), systemic lupus erythematosus (SLE),chronic polyarthritis, Basedow's disease, autoimmune forms of chronichepatitis, colitis ulcerosa, type I allergy diseases, type II allergydiseases, type III allergy diseases, type IV allergy diseases,fibromyalgia, hair loss, Bechterew's disease, Crohn's disease,Myasthenia gravis, neurodermitis, Polymyalgia rheumatica, progressivesystemic sclerosis (PSS), psoriasis, Reiter's syndrome, rheumaticarthritis, psoriasis, vasculitis, etc, or type II diabetes. While theexact mode as to why the immune system induces an immune reactionagainst autoantigens has not been elucidated so far, there are severalfindings with regard to the etiology. Accordingly, the autoreaction maybe due to a T-Cell Bypass. A normal immune system requires theactivation of B-cells by T-cells before the former can produceantibodies in large quantities. This requirement of a T-cell can beby-passed in rare instances, such as infection by organisms producingsuper-antigens, which are capable of initiating polyclonal activation ofB-cells, or even of T-cells, by directly binding to one subunit ofT-cell receptors in a non-specific fashion. Another explanation deducesautoimmune diseases from a molecular mimicry. An exogenous antigen mayshare structural similarities with certain host antigens; thus, anyantibody produced against this antigen (which mimics the self-antigens)can also, in theory, bind to the host antigens and amplify the immuneresponse. The most striking form of molecular mimicry is observed inGroup A beta-haemolytic streptococci, which shares antigens with humanmyocardium, and is responsible for the cardiac manifestations ofrheumatic fever. The present invention allows therefore provision of apharmaceutical composition containing an autoantigen (as protein, mRNAor DNA encoding for a autoantigen protein) and a nucleic acid of theinvention which typically allows the immune system to be desensitized.

The invention relates also to the use of at least one inventive nucleicacid molecule of either formula (I), (Ia), (II), (IIa), (IIb), (IIIc)and/or (IIIb) according to the invention as defined above in thepreparation of a pharmaceutical composition according to the inventionor of a vaccine according to the invention for the treatment ofindications described hereinbefore, for example for the treatment of thementioned tumour, autoimmune diseases, allergies and infectiousdiseases. Alternatively, the invention includes the (therapeutic) use ofat least one nucleic acid molecule of either formula (I), (Ia), (II),(IIa), (IIb), (IIIc) and/or (IIIb) according to the invention as definedabove, for the treatment of tumour or infectious diseases, as describedhereinbefore.

Likewise included in the present invention are kits, e.g. kit of parts,(each part) containing at least one nucleic acid molecule of eitherformula (I), (Ia), (II), (IIa), (IIb), (IIIc) and/or (IIIb) according tothe invention, and/or a pharmaceutical composition according to theinvention and/or a vaccine according to the invention as well as,optionally, technical instructions for use with information on theadministration and dosage of the at least one nucleic acid molecule ofeither formula (I), (Ia), (II), (IIa), (IIb), (IIIc) and/or (IIIb)according to the invention as defined above, and/or of thepharmaceutical composition according to the invention and/or of thevaccine according to the invention.

Methods of treating a disorder or disease selected from the groupconsisting of cancer diseases, infectious diseases, autoimmune diseasesand allergies by administering to a patient in need thereof apharmaceutically effective amount of a nucleic acid molecule accordingto the invention.

FIGURES

The following Figures are intended to illustrate the invention further.They are not intended to limit the subject matter of the inventionthereto.

FIG. 1: shows the TNFα inducing capacity of DOTAP formulated RNAsaccording to formula (I). PBMCs were seeded at a density of2*10⁵/well/200 μl Medium and stimulated with RNA (4 μg/ml) formulatedwith DOTAP (12 μg/ml) for 20 h. A TNFα-ELISA was then performed withcell free supernatants. As can be seen in FIG. 1, secretion of TNFα issignificantly induced by the inventive nucleic acids according toformula (I), particularly by mRNA sequences according to SEQ ID NOs: 114to 119 inventive nucleic acids according to formula (I) as definedabove, i.e. mRNA sequences according to SEQ ID NOs: 114 to 119 (SEQ IDNO: 114 (R820/(N100)₂), SEQ ID NO: 115 (R719/(N100)₅), SEQ ID NO: 116(R720/(N100)₁₀), SEQ ID NO: 117 (R821/(N40T20N40)₂), SEQ ID NO: 118(R722/(N40T20N40)₅), and SEQ ID NO: 119 (R723/(N40T20N40)₁₀)) andcontrols G₂U₂₀G₂ (GGUUUUUUUUUUUUUUUUUUUUGG) (SEQ ID NO: 125), Seq. U₂₁:UUUUUUUUUUUUUUUUUUUUU (SEQ ID NO: 126) (Phosphodiester) and Poly(U)(Sigma, 800-1000 kDa).

FIG. 2: shows the IFNα inducing capacity of DOTAP formulated RNAsaccording to formula (I). PBMCs were seeded at a density of2*10⁵/well/200 μl Medium and stimulated with RNA (2 μg/ml) formulatedwith DOTAP (12 μg/ml) for 20 h. An IFNα-ELISA was then performed withcell free supernatants. As can be seen in FIG. 2, secretion of IFNα issignificantly induced by the inventive nucleic acids according toformula (I), particularly by mRNA sequences according to SEQ ID NOs: 114to 119 inventive nucleic acids according to formula (I) as definedabove, i.e. mRNA sequences according to SEQ ID NOs: 114 to 119 (SEQ IDNO: 114 (R820/(N100)₂), SEQ ID NO: 115 (R719/(N100)₅), SEQ ID NO: 116(R720/(N100)₁₀), SEQ ID NO: 117 (R821/(N40T20N40)₂), SEQ ID NO: 118(R722/(N40T20N40)₅), and SEQ ID NO: 119 (R723/(N40T20N40)₁₀)) andcontrols G₂U₂₀G₂ (GGUUUUUUUUUUUUUUUUUUUUGG) (SEQ ID NO: 125), Seq. U₂₁:UUUUUUUUUUUUUUUUUUUUU (SEQ ID NO: 126) (Phosphodiester) and Poly(U)(Sigma, 800-1000 kDa).

EXAMPLES

The following Examples are intended to illustrate the invention further.They are not intended to limit the subject matter of the inventionthereto.

1. Synthesis of Exemplary Nucleic Acids of Either Formula (I), (Ia),(II), (IIa), (IIb), (IIIa) and/or (IIIb) According to the Invention

-   -   RNA oligonucleotides, as examples of the nucleic acid of the        general formula (I), (Ia), (II), (IIa), (IIb), (IIIa) and/or        (IIIb) according to the invention, were prepared by automatic        solid-phase synthesis by means of phosphoramidite chemistry        (including sequences according to SEQ ID NOs: 84-85 (formula        (I)), SEQ ID NOs: 86-87 (formula (Ia)), SEQ ID NOs: 88-94        (formulas (II), (IIa) and (IIb)), and SEQ ID NOs: 107-108        (formulas (IIIa) and (IIIb))). In each case the RNA-specific        2′-hydroxyl groups of the nucleotides were protected with TBDMS        protecting groups. In the synthesis of phosphorothioates,        Beaucage reagent was used for the oxidation. The cleavage of        carrier material and of the base-labile protecting groups was        carried out with methylamine, and the cleavage of the TBDMS        protecting group was effected with triethylamine hydrofluoride.    -   The crude product was purified by means of HPLC either by        ion-pair chromatography, by ion-exchange chromatography or by a        combination of the two methods, desalinated and dried. The        product was checked for purity and correct base composition by        mass spectrometry.    -   According to an alternative way, the above sequences were        prepared by in vitro translation based on DNA vectors or        oligonucleotide sequences carrying the inventive sequences.

2. In Vitro Immunostimulation with Exemplary Nucleic Acids of EitherFormula (I), (Ia), (II), (IIa), (IIb), (IIIa) and/or (IIIb) According tothe Invention

-   -   a) For the stimulation of mouse BDMCs (bone marrow derived        dendritic cells), 3 μl of oligofectamine were mixed with 30 μl        of FCS-free IMDM medium (BioWhittaker, catalogue no. BE12-722F)        and incubated at room temperature for 5 minutes. 6 μg of a        nucleic acid according to SEQ ID NOs: 84-94 and 107-108 (each        type of nucleic acid forming a single experiment), respectively,        in the form of RNA, was mixed with 60 μl of FCS-free IMDM and        mixed with oligofectamine/IMDM, and incubated for 20 minutes at        room temperature. 33 μl of this mixture were then placed for        cultivation overnight in a well of a 96-well microtitre culture        plate which contained 200,000 mouse BDMCs in 200 μl of FCS-free        IMDM medium. After 4 hours, 100 μl of IMDM containing 20% FCS        were added and, after 16 hours' co-incubation, the supernatant        was removed and tested for interleukin-6 (IL-6) and        interleukin-12 (IL-12) by a cytokine ELISA. Comparison tests        were carried out analogously to the above sequences using the        immunostimulating uncapped wild-type mRNA of beta-galactosidase        (lacZ), complexed with protamine.        -   It was possible to show that the nucleic acids of formulas            (I), (Ia), (II), (IIa), (IIb), (IIIc) and/or (IIIb)            according to the invention, present in the form of RNA, in            particular the sequences according to the invention of SEQ            ID NOs: 84-94 and 107-108, have good immunostimulating            properties for stimulation of an innate immune response.    -   b) Human PBMCs were obtained via a Ficoll density gradient and        cultivation overnight in X-VIVO-15 medium (BioWhittaker,        catalogue no. BE04-418Q), which contained 1% glutamine and 1%        penicillin in the presence of 10 μg/ml of the nucleic acids of        either formula (I), (Ia), (II), (IIa), (IIb), (IIIc) and/or        (IIIb) according to the invention in the form of RNA, in        particular of the sequences according to the invention of SEQ ID        NOs: 84-94 and 107-108 (each type of nucleic acid forming a        single experiment).        -   For stimulation, 3 μl of oligofectamine were mixed with 30            μl of X-VIVO-15 medium (BioWhittaker, catalogue no.            BE04-418Q) and incubated at room temperature for 5 minutes.            6 μg of the nucleic acids of either formula (I), (Ia), (II),            (IIa), (IIb), (IIIc) and/or (IIIb) according to the            invention in the form of RNA, in particular the sequences            according to the invention SEQ ID NOs: 84-94 and 107-108            (each type of nucleic acid in a single experiment),            respectively, were mixed with 60 μl of X-VIVO-15 medium            (BioWhittaker, catalogue no. BE04-418Q) and, mixed with            oligofectamine/X-VIVO medium, incubated for 20 minutes at            room temperature. 33 μl of this mixture were then placed for            cultivation overnight in a well of a 96-well microtitre            culture plate which contained 200,000 PBMCs in 200 μl of            X-VIVO-15 medium (BioWhittaker, catalogue no. BE04-418Q).            After co-incubation for 16 hours, the supernatant was            removed and tested for interleukin-6 (IL-6) and            interleukin-12 (IL-12) and TNFα by means of a            cytokine-ELISA. Comparison tests were carried out            analogously to the sequences according to the invention (see            above) with the immunostimulating oligo RNA40            (5′-GCCCGUCUGUUGUGUGACUC-3′, SEQ ID NO: 113).        -   It was possible to show that the inventive nucleic acids in            the form of RNA, in particular having the sequences            according to the invention either formula (I), (Ia), (II),            (IIa), (IIb), (IIIa) and/or (IIIb) according to the            invention as defined above have good immunostimulating            properties.

3. In Vivo Immunostimulation with Exemplary Nucleic Acids of EitherFormula (I), (Ia), (II), (IIa), (IIb), (IIIa) and/or (IIIb) According tothe Invention—Use as Adjuvant

-   -   BALB/c mice (5 per group) were injected with beta-galactosidase        protein and with an adjuvant (as defined herein) on days 0        and 10. The mice were sacrificed on day 20 and the blood serum        was used for an antibody test against beta-galactosidase protein        by means of ELISA, and the IL-6, IL-12 and TNF-alpha values were        determined analogously to the above-described in vitro cultures.

4. Stimulation of Human Cells with an Adjuvant According to theInvention in the Form of a Nucleic Acid Molecule of Either Formula (I),(Ia), (II), (IIa), (IIb), (IIIa) and/or (IIIb)

-   -   a) In order to determine the immunogenic activity of nucleic        acids of either formula (I), (Ia), (II), (IIa), (IIb), (IIIa)        and/or (IIIb) according to the invention as defined above in the        form of adjuvants, particularly of nucleic acids containing a        sequence according to SEQ ID NOs: 84-94 and 107-108 (each type        of nucleic acid again forming a single experiment) were        co-incubated with human cells. To this end, human PBMC cells,        for example, were co-incubated for 16 hours in X-VIVO-15 medium        (BioWhittaker, catalogue no. BE04-418Q), enriched with 2 mM        L-glutamine (BioWhittaker), 10 U/ml penicillin (BioWhittaker)        and 10 μg/ml streptomycin, with 10 μg/ml of RNA (mRNA coding for        β-galactosidase and optionally with 10 μg/ml protamine. The        supernatants were removed and the release of IL-6 and TNFalpha        was analysed by means of ELISA.    -   b) In a further experiment, the release of TNF-alpha by human        PBMC cells was determined after stimulation with inventive        nucleic acids of either formula (I), (Ia), (II), (IIa), (IIb),        (IIIa) and/or (IIIb) according to the invention (SEQ ID NOs:        84-94 and 107-108, each type of nucleic acid in a single        experiment, see above) and also adjuvants used according to the        invention.        -   To that end, human PBMC cells were co-incubated for 16 hours            with 10 μg/ml said inventive nucleic acids in X-VIVO 15            medium (BioWhittaker), enriched with 2 mM L-glutamine            (BioWhittaker), 10 U/ml penicillin (BioWhittaker) and 10            μg/ml streptomycin. The supernatants were removed and            analysed by means of ELISA.

5. Secretion of TNFφ and IFN-α in Human PBMCs

For this experiments, several inventive nucleic acids according toformula (I) as defined above, i.e. mRNA sequences according to SEQ IDNOs: 114 to 119, were formulated with DOTAP (Roche).

The inventive nucleic acid sequences used in the experiment were

-   -   SEQ ID NO: 114 (R820/(N100)₂);    -   SEQ ID NO: 115 (R719/(N100)₅);    -   SEQ ID NO: 116 (R720/(N100)₁₀);    -   SEQ ID NO: 117 (R821/(N40T20N40)₂);    -   SEQ ID NO: 118 (R722/(N40T20N40)₅); and    -   SEQ ID NO: 119 (R723/(N40T20N40)₁₀).

Human PBMCs were then stimulated with the formulated RNAs at aconcentration of 8 μg/ml and 12 μg/ml DOTAP for 20 hours. TheSupernatants were then investigated for the secretion of TNFα and IFN-αusing a matched-paired ELISA.

For the experiment, human PBMCs were obtained via a Ficoll densitygradient and cultivation for 20 hours in X-VIVO-15 medium (BioWhittaker,catalogue no. BE04-418Q), which contained 1% glutamine and 1% penicillinin the presence of 2 or 4 □g/ml of the above nucleic acids for IFNα orTNFα stimulation respectively. For formulation and stimulation, 3 or 6μg RNA in HBS buffer were transferred to a vial containing 18 μgN-[4-(2,3-Dioleoyloxy)propyl]-N,N,Ntrimethylammonium methylsulfate(DOTAP) (Roche Diagnostics, catalogue no. 11 811 177 001) in HBS bufferand carefully mixed by gently pipetting the mixture several times. Thetransfection mixture was incubated for 15 min at 15-25° C. 1 volume ofthe DOTAP/nucleic acid mixture was then gently diluted with 7.3 volumesof X-Vivo medium. 100 μl of this mixture were then placed forcultivation overnight in a well of a 96-well microtitre culture platewhich contained 2*10⁵ PBMCs in 100 μl of X-VIVO-15 medium (BioWhittaker,catalogue no. BE04-418Q). After coincubation for 20 hours, thesupernatant was removed and tested for IFNα and TNFφ□ by means of acytokine-ELISA. Comparison tests were carried out analogously to thesequences according to the invention (see above) with theimmunostimulating oligo G₂U₂₀G₂ (Phosphothioat-modifed), Poly(U) (Sigma,Taufkirchen, Germany) and the oligo U₂₁ (Phophodiester).

The results are shown in FIGS. 1 and 2. FIG. 1 shows the TNFα inducingcapacity of DOTAP formulated RNAs. PBMCs were seeded at a density of2*10⁵/well/200 μl Medium and stimulated with RNA (4 μg/ml) formulatedwith DOTAP (12 μg/ml) for 20 h. A TNFα-ELISA was then performed withcell free supernatants. FIG. 2 shows the IFNα inducing capacity of DOTAPformulated RNAs. PBMCs were seeded at a density of 2*10⁵/well/200 μlMedium and stimulated with RNA (2 μg/ml) formulated with DOTAP (12μg/ml) for 20 h. An IFNα-ELISA was then performed with cell freesupernatants.

As can be seen in FIG. 1 and FIG. 2, both secretion of TNFα and IFNα issignificantly induced by the inventive nucleic acids according toformula (I), particularly by mRNA sequences according to SEQ ID NOs: 114to 119 inventive nucleic acids according to formula (I) as definedabove, i.e. mRNA sequences according to SEQ ID NOs: 114 to 119 (SEQ IDNO: 114 (R820/(N100)₂), SEQ ID NO: 115 (R719/(N100)₅), SEQ ID NO: 116(R720/(N100)₁₀), SEQ ID NO: 117 (R821/(N40T20N40)₂), SEQ ID NO: 118(R722/(N40T20N40)₅), and SEQ ID NO: 119 (R723/(N40T20N40)₁₀)) versuscontrol sequences G₂U₂₀G₂ (Phosphothioat-modifed), Poly(U) (Sigma,Taufkirchen, Germany) and the oligo U₂₁ (Phophodiester).

ADVANTAGES OF THE INVENTION

A nucleic acid of the general formula (I), (Ia), (II), (IIa), (IIb),(IIIc) and/or (IIIb) according to the invention may be used asimmunostimulating agent as such for stimulating the innate immune systemof a patient to be treated. This immunostimulating property may well beenhanced by the addition of other compounds known in the art as activelystimulating the innate immune response to the inventive nucleic acids,e.g. by lipid modification or addition of additional adjuvants. Theinventive nucleic acids as defined herein, particularly those accordingto formula (I) comprising the structure (N_(u)G_(l)X_(m)G_(n)N_(v))_(a),or of derivatives thereof, exhibit a significant better amplification inbacteria, e.g. E. coli. It is furthermore particularly advantageous, ifthe inventive nucleic acid (N_(v)G_(l)X_(m)G_(n)N_(u))_(a) of formula(I), or of derivatives thereof, is a partially double-stranded nucleicacid molecule or a mixture of a single-stranded and a double-strandednucleic acid molecule, since such a (partially double-stranded)inventive nucleic acid molecule according to formula (I) (or of formula(Ia), (II) (IIa), (IIb), (IIIc) and/or (IIIb)), can positively stimulatethe innate immune response in a patient to be treated by addressing thePAMP—(pathogen associated molecular pattern) receptors forsingle-stranded RNA (TLR-7 and TLR-8) as well as the PAMP-receptors fordouble-stranded RNA (TLR-3, RIG-I and MDA-5). Receptors TLR-3, TLR-7 andTLR-8 are located in the endosome and are activated by RNA taken up bythe endosome. In contrast, RIG-I and MDA-5 are cytoplasmic receptors,which are activated by RNA which was directly taken up into thecytoplasm or which has been released from the endosomes (endosomalrelease or endosomal escape). Accordingly, a partially double-strandedinventive nucleic acid (N_(u)G_(l)X_(m)G_(n)N_(v))_(a) of formula (I)(or of derivatives thereof, e.g. (a partially double-stranded) inventivenucleic acid molecule according to formula (Ia), (II) (IIa), (IIb),(IIIc) and (IIIb) as defined herein)) is capable of activating differentsignal cascades of immunostimulation and thus leads to an increasedinnate immune response or enhances such a response significantly. Afurther advantage of the invention is the high induction of theantiviral cytokine IFNalpha which is preferred in stimulation of theinnate immune system. An often underestimated limitation of generallyaccepted immunostimulating nucleic acids (e.g. poly A:U and poly I:C) isthe undefined structure of them which results in regulatoryrestrictions.

1-25. (canceled)
 26. An isolated nucleic acid molecule comprising atleast 100 contagious nucleotides of SEQ ID NO: 114-119 or the DNA codingsequence thereof, provided that: the guanosine (guanine), the uridine(uracil), the adenosine (adenine), the thymidine (thymine), or thecytidine (cytosine) positions of the nucleotide (nucleoside) sequence ofthe nucleic acid molecule may be substituted with an analogue ofnucleotides (nucleosides) selected from 1-methyl-adenosine (adenine),2-methyl-adenosine (adenine), 2-methylthio-N6-isopentenyl-adenosine(adenine), N6-methyl-adenosine (adenine), N6-isopentenyl-adenosine(adenine), 2-thio-cytidine (cytosine), 3-methyl-cytidine (cytosine),4-acetyl-cytidine (cytosine), 2,6-diaminopurine, 1-methyl-guanosine(guanine), 2-methyl-guanosine (guanine), 2,2-dimethyl-guanosine(guanine), 7-methyl-guanosine (guanine), inosine, 1-methyl-inosine,dihydro-uridine (uracil), 4-thio-uridine (uracil),5-carboxymethylaminomethyl-2-thio-uridine (uracil),5-(carboxyhydroxylmethyl)-uridine (uracil), 5-fluoro-uridine (uracil),5-bromo-uridine (uracil), 5-carboxymethylaminomethyl-uridine (uracil),5-methyl-2-thio-uridine (uracil), N-uridine (uracil)-5-oxyacetic acidmethyl ester, 5-methylaminomethyl-uridine (uracil),5-methoxyaminomethyl-2-thio-uridine (uracil),5′-methoxycarbonylmethyl-uridine (uracil), 5-methoxy-uridine (uracil),uridine (uracil)-5-oxyacetic acid methyl ester, uridine(uracil)-5-oxyacetic acid (v), queosine, beta-D-mannosyl-queosine,wybutoxosine, and inosine; phosphate moieties may be substituted withphosphoramidates, phosphorothioates, peptide nucleotides, ormethylphosphonates; and ribose components may be substituted withdeoxyribose.
 27. The isolated nucleic acid molecule of claim 26,comprising at least 150 contagious nucleotides of SEQ ID NO: 114-119.28. The isolated nucleic acid molecule of claim 27, comprising at least200 contagious nucleotides of SEQ ID NO: 114-119.
 29. The isolatednucleic acid molecule of claim 26, comprising at least 200 contagiousnucleotides of SEQ ID NO: 114-116.
 30. The isolated nucleic acidmolecule of claim 29, comprising at least 200 contagious nucleotides ofSEQ ID NO:
 114. 31. The isolated nucleic acid molecule of claim 30,comprising the sequence of SEQ ID NO:
 114. 32. The isolated nucleic acidmolecule of claim 26, comprising at least 200 contagious nucleotides ofSEQ ID NO: 117-119.
 33. The isolated nucleic acid molecule of claim 32,comprising at least 200 contagious nucleotides of SEQ ID NO:
 117. 34.The isolated nucleic acid molecule of claim 33, comprising the sequenceof SEQ ID NO:
 117. 35. The isolated nucleic acid molecule of claim 26,wherein the nucleic acid molecule is RNA.
 36. The isolated nucleic acidmolecule of claim 26, wherein the nucleic acid molecule issingle-stranded.
 37. The isolated nucleic acid molecule of claim 26,wherein the nucleic acid molecule is double-stranded or partiallydouble-stranded.
 38. The isolated nucleic acid molecule of claim 26,wherein the nucleic acid molecule is linear.
 39. The isolated nucleicacid molecule of claim 26, wherein the nucleic acid molecule iscomplexed with a lipid, polycationic compound, or polycationicpolypeptide.
 40. The isolated nucleic acid molecule of claim 26, whereinthe nucleic acid molecule does not comprise a nucleoside analogue. 41.The isolated nucleic acid molecule of claim 26, comprising at least 200contagious nucleotides of SEQ ID NO: 114-119 or the DNA coding sequencethereof, provided that: the guanosine (guanine), the uridine (uracil),the adenosine (adenine), the thymidine (thymine), or the cytidine(cytosine) positions of the nucleotide (nucleoside) sequence of thenucleic acid molecule may be substituted with an analogue of nucleotides(nucleosides) selected from 1-methyl-adenosine (adenine),2-methyl-adenosine (adenine), 2-methylthio-N6-isopentenyl-adenosine(adenine), N6-methyl-adenosine (adenine), N6-isopentenyl-adenosine(adenine), 2-thio-cytidine (cytosine), 3-methyl-cytidine (cytosine),4-acetyl-cytidine (cytosine), 2,6-diaminopurine, 1-methyl-guanosine(guanine), 2-methyl-guanosine (guanine), 2,2-dimethyl-guanosine(guanine), 7-methyl-guanosine (guanine), inosine, 1-methyl-inosine,dihydro-uridine (uracil), 4-thio-uridine (uracil),5-carboxymethylaminomethyl-2-thio-uridine (uracil),5-(carboxyhydroxylmethyl)-uridine (uracil), 5-fluoro-uridine (uracil),5-bromo-uridine (uracil), 5-carboxymethylaminomethyl-uridine (uracil),5-methyl-2-thio-uridine (uracil), N-uridine (uracil)-5-oxyacetic acidmethyl ester, 5-methylaminomethyl-uridine (uracil),5-methoxyaminomethyl-2-thio-uridine (uracil),5′-methoxycarbonylmethyl-uridine (uracil), 5-methoxy-uridine (uracil),uridine (uracil)-5-oxyacetic acid methyl ester, uridine(uracil)-5-oxyacetic acid (v), queosine, beta-D-mannosyl-queosine,wybutoxosine, and inosine; phosphate moieties may be substituted withphosphoramidates, phosphorothioates, peptide nucleotides, ormethylphosphonates; and ribose components may be substituted withdeoxyribose.
 42. The isolated nucleic acid molecule of claim 41, whereinthe nucleic acid molecules is RNA.
 43. The isolated nucleic acidmolecule of claim 41, wherein the nucleic acid molecule does notcomprise a nucleoside analogue.
 44. A pharmaceutical compositioncomprising the nucleic acid molecule according to claim 26 and apharmaceutically acceptable carrier.
 45. The pharmaceutical compositionof claim 44, further comprising an adjuvant.
 46. The pharmaceuticalcomposition of claim 44, further comprising an antigen.
 47. Thepharmaceutical composition of claim 44, wherein the antigen is a RNAencoding polypeptide antigen.
 48. The pharmaceutical composition ofclaim 44, wherein the antigen is a polypeptide antigen.